Modified capsid proteins for enhanced delivery of parvovirus vectors

ABSTRACT

This invention relates to modified parvovirus capsid proteins with enhanced transduction efficiency, viral vectors comprising the same, and methods of using the same for delivery of nucleic acids to a cell or a subject.

STATEMENT OF PRIORITY

This application is a 35 U.S.C. § 371 national phase application ofPCT/US2016/066466, filed Dec. 14, 2016, which claims the benefit of U.S.Provisional Application Ser. No. 62/266,941, filed Dec. 14, 2015, theentire contents of each of which are incorporated by reference herein.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under Grant Nos.AI080726, DK084033, HL112761, AI072176, AR064369, and GM007050 awardedby the National Institutes of Health. The government has certain rightsin the invention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 5470-710_ST25.txt, 9,958 bytes in size, generated onJun. 11, 2018 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

This invention relates to modified parvovirus capsid proteins withenhanced transduction efficiency, viral vectors comprising the same, andmethods of using the same for delivery of nucleic acids to a cell or asubject.

BACKGROUND OF THE INVENTION

Various serotypes of recombinant adeno-associated virus (rAAV) arecurrently being utilized in clinical trials (e.g., rAAV1 for lipoproteinlipase deficiency, rAAV2 for Leber Congenital Amaurosis, rAAV8 forhemophilia). Of these, rAAV1 is considered the best for intramuscular(i.m.) delivery, whether treating muscle disorder directly (e.g.,muscular dystrophy) or for conditions that can benefit from thesecretion of a therapeutic protein into the bloodstream (e.g., α-1antitrypsin (AAT) deficiency). A major limitation of this approach hasbeen sub therapeutic levels of transgene expression, despite theadministration of high doses of rAAV. For example, while results fromrAAV-AAT clinical trials demonstrate dose dependent increases in serumlevels of AAT following i.m. injection, in high dose cohorts only ˜3% oftarget AAT expression was achieved. As this cohort required 100 i.m.injections of 1.35 mL each, increasing the dose to the degree necessaryfor disease correction may not be feasible. Similar observations havebeen documented for the recently approved rAAV1-based pharmaceutical,Glybera. For rAAV gene augmentation therapy to become a practical choicein cases such as these, improving transduction efficiency in muscletissue is essential.

Efforts towards increasing transgene expression can be divided intoendogenous approaches, wherein the rAAV capsid and transgene themselvesare modified to enhance transduction, and/or exogenous approaches,wherein supplementary therapeutics are delivered concurrent to rAAVadministration. While exogenous approaches such as immune suppressionhave improved transduction, the process of integrating new discoveriesof rAAV biology into clinical trial design has been the most successfultactic. This is best illustrated with the development of numerousnaturally occurring AAV serotypes, and their preferential tissuetransduction profiles. This enables correct pairing of AAV serotype totarget organ (e.g., clinical trials utilizing i.m. delivery of rAAVbecame demonstrably more successful when rAAV1 was employed, as opposedto the historically utilized rAAV2). Additionally, serotype-comparativebiological and structural analyses have facilitated directed evolutionand rational engineering of rAAV, leading to the development of nextgeneration translational vectors.

The capsid structure of most common rAAV serotypes has been resolved.Each contains 60 repeating monomers, comprised of a conserved β-barrelcore interspersed with large loops that form the topology of the capsidsurface. By comparing the structures of rAAV2 and rAAV4, the leasthomologous serotypes, a total of nine variable regions (VRs; VRI toVRIX) were defined. The amino acid content of the VRs contributesdistinct phenotypes to each serotype, such as receptor binding,antigenic reactivity and transduction efficiency. Comparing the VRs ofrAAV2 (the prototypical rAAV) with more efficient muscle transducers,such as rAAV1, led to the first engineered rAAV capsid to enter clinicaltrial. rAAV2.5 consists of an rAAV2 capsid engrafted with five aminoacids from rAAV1, isolated for their contribution to rAAV1's efficiencyin muscle. Follow-up studies revealed that only a single amino acidchange was needed to markedly enhance rAAV2 efficiency in muscle;namely, insertion of various amino acids following position 264, therebycreating a de novo position 265. Continued pre-clinical studies havesuggested that the translatability of rAAV2 is limited by the superiorefficiency of serotypes such as rAAV1 and rAAV6, as well as the highprevalence of rAAV2 neutralizing antibodies within the population. These“next generation” serotypes have been augmented further for efficienttransduction by modulation of surface amino acids on the capsidbackbone.

As rAAV1 has become the top choice for musculoskeletal application,being used in 67% of i.m.-based clinical trials overall and 86% in thepast 5 years, improving rAAV1 efficiency is timely to clinicaltranslation. The present invention provides modified capsid proteinsthat have improved characteristics and are suitable for generatingvectors with a wide variety of uses, including gene therapy.

SUMMARY OF THE INVENTION

This study dissected rAAV capsid architecture to develop an engineeringstrategy designed to improve muscle transduction. Contrary to rAAV2 265insertion mutants, the deletion of position 265 from the rAAV1 capsidprovided the highest level of enhancement for both transgene deliveryand expression in muscle tissue. Furthermore, through homology modelingand mutational analysis, two regions of the capsid were identified thatappear to work together allosterically to control transductionefficiency in rAAV6 and rAAV2. The results provide a mechanism ofregional destabilization in the VR1 loop due to the destruction ofhydrogen bonding patterns. This discovery allowed for rational mutationof these additional serotypes in order to enhance transductionefficiency by at least an order of magnitude in each case. Furthermore,expression of the clinical AAT transgene in the chimera rAAV serotypesincreased expression by up to 12.5-fold over parental rAAV1, supportingthe use of these constructs in clinical trials. This study validates arational design approach using structural modeling and moleculardissection of the rAAV capsid for improved delivery reagents bettersuited for translational studies.

One aspect of the invention relates to a parvovirus capsid proteincomprising a capsid protein amino acid sequence from an AAV serotype orany other parvovirus with an icosahedral capsid structure of T=1,wherein the variable region 1 (VR1) loop comprising amino acid residues258 to 272 of AAV1 capsid protein or the corresponding amino acidresidues from another AAV or parvovirus capsid protein is modified bydeletion and/or substitution of one or more amino acid residues to causeregional destabilization within the loop due to the targeted destructionof hydrogen bonding patterns orchestrated by the residues, wherein thecapsid protein comprising the modification provides to a virus vectorcomprising the capsid protein increased transduction efficiency relativeto a virus vector comprising a capsid protein that does not contain themodification.

An additional aspect of the invention relates to the capsid protein ofthe invention, wherein the capsid protein comprises an amino acidsequence from an AAV serotype or other parvovirus that binds to heparinsulfate, wherein one or more amino acid residues that mediate binding ofthe capsid protein to heparin sulfate are substituted and/or deleted,wherein binding of the capsid protein to heparin sulfate issubstantially reduced.

A further aspect of the invention relates to an AAV capsid proteincomprising an amino acid sequence from an AAV3a, AAV3b, AAV6, or AAV8serotype, wherein one or more amino acid residues that mediate bindingof the capsid protein to heparin sulfate are substituted and/or deleted,wherein binding of the capsid protein to heparin sulfate issubstantially reduced.

An additional aspect of the invention relates to an AAV capsid proteincomprising an amino acid sequence from an AAV2, AAV3a, or AAV3bserotype, wherein the capsid protein comprises an insertion of one ormore amino acid residues immediately following residue 264 of AAV2capsid protein or the corresponding residue of AAV3a or AAV 3b capsidprotein, and one or more amino acid residues that mediate binding of thecapsid protein to heparin sulfate are substituted and/or deleted,wherein binding of the capsid protein to heparin sulfate issubstantially reduced; wherein the capsid protein provides to a virusvector comprising the capsid protein increased transduction efficiencyrelative to a virus vector comprising an unmodified capsid protein.

A further aspect of the invention relates to a polynucleotide encodingthe capsid protein of the invention, a parvovirus capsid comprising thecapsid protein of the invention, a virus vector comprising the capsidprotein of the invention, and a pharmaceutical composition comprisingthe virus vector of the invention.

Another aspect of the invention relates to a method of delivering anucleic acid to a cell, the method comprising contacting the cell withthe virus vector or the pharmaceutical composition of the inventionunder conditions sufficient for the nucleic acid to enter the cell.

A further aspect of the invention relates to method of delivering anucleic acid to a subject, the method comprising administering to thesubject the virus vector or the pharmaceutical composition of theinvention.

Another aspect of the invention relates to method of delivering anucleic acid to a subject, the method comprising administering to thesubject a cell that has been contacted with the virus vector or thepharmaceutical composition of the invention under conditions sufficientfor the nucleic acid to enter the cell.

An additional aspect of the invention relates to a method of producing arecombinant parvovirus particle, comprising providing to a cellpermissive for parvovirus replication: (a) a recombinant parvovirustemplate comprising (i) a heterologous nucleic acid, and (ii) at leastone inverted terminal repeat; and (b) a polynucleotide comprisingreplication protein coding sequence(s) and sequence(s) encoding thecapsid protein of the invention; under conditions sufficient for thereplication and packaging of the recombinant parvovirus template;whereby recombinant parvovirus particles are produced in the cell.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show in vivo characterization of rAAV1 position 265 mutantsfollowing i.m. injection. (A) Mice were administered 1e10 vg into theGC. Luciferase transgene expression was visualized and quantified usinglive animal bioluminescent imaging at 7 dpi. Units displayed are countsper minute over region of interest (CPM/ROI). For visualizationpurposes, rAAV1 is shown on a different scale than the 265 mutants. Thisdoes not affect image quantitation. For ex vivo quantification oftransduction phenotype (B), injected muscle was harvested and lysed, andluciferase assay performed on tissue lysate. Measurements depicted asrelative light units normalized to mg total protein (RLU/mg protein).(C) Viral transgene copy numbers per cell (vg/cg) were measured usingqPCR. (D) A time course of expression kinetics was performed on rAAV1and rAAV1/T265del following injection of 1e10 vg. All data representsn=4 per group; in panel (A) one representative image is displayed pergroup.

FIGS. 2A-2C show the phenotype of 265 deletion mutant in rAAV6. (A)Luciferase transgene expression 7 dpi of 1e10 vg of either rAAV6 orrAAV6/T265del. (B) Alignment of the VR1 capsid region of rAAV1 (lightgrey) and rAAV6 (dark grey). Residue 265 is highlighted in dark blue onrAAV1 and in yellow on rAAV6. Image generated in PyMol using availablecrystallographic coordinates for these capsids. (C) Transgene expressionfollowing injection of rAAV1→rAAV6 point mutants into mouse GC. Datarepresented as fold difference relative to measured expression of rAAV6.All data represents n=4 per group.

FIGS. 3A-3D show the visualization of amino acid positions 265 and 531on the rAAV6 capsid. Pymol depiction of the crystallographic coordinatesof the rAAV6 capsid (A), with each icosahedral axis of symmetry labeled.(B) Close up of the 3-fold axis of symmetry, with residues 265 and 531colored purple and orange, and denoted by arrows. (C) Close up of the5-fold axis of symmetry, with residues 265 and 531 colored purple andorange, and denoted by arrows. (D) Close up of the 2-fold axis ofsymmetry, with residues 265 and 531 colored purple and orange, anddenoted by arrows.

FIGS. 4A-4C show the effect of capsid:heparin interactions on thetransduction phenotype of 265 mutation. (A) Transgene expression 7 dpiof rAAV2 capsid mutants into the GC. (B) Pymol depiction of the rAAV6capsid with residues T265 (purple), K531 (orange) and R585 (blue)denoted, in the context of the 3-fold axis of symmetry. Dashed circlerepresents general outline of heparin binding footprint. (C) Transgeneexpression of rAAV2 and rAAV1 constructs injected into GC followingpreincubation in either a saline or heparin sulfate solution. All datarepresents n=4 per group.

FIGS. 5A-5D show heparin affinity chromatography elution profiles ofrAAV2 and rAAV6 constructs used in this study. rAAV2 (A, B) and rAAV6(C, D) constructs used in this study were incubated withheparin-conjugated agarose beads and then eluted from beads usingincreasingly stringent NaCl washes. Elution profiles were collected byquantifying the number of viral genomes present in each eluted fractionusing RT-qPCR. Experiments were repeated in triplicate; onerepresentative chromatogram is shown per group.

FIG. 6 shows transgene expression of VR1 deletion scanning mutants inthe rAAV1 capsid. Single deletion mutations were created in the VR1 loopof the rAAV1 capsid, and constructs were injected into mouse GC at adose of 1e10vg. Transgene expression is depicted as fold differencerelative to that measured following injection of rAAV1. Cartoon abovegraph depicts residues that were mutated for this study in light grey,with position 265 depicted in dark grey. The location of N- andC-terminal beta sheets surrounding VR1 is also shown. Data isrepresentative of n=4 per group.

FIG. 7 shows serum concentration of hAAT following i.m. injection ofrAAV1 and rAAV6 constructs. rAAV1 and rAAV6 capsid packaging an hAATtransgene were injected into GC muscle at a dose of 1e10vg. 5 weeks postinjection, serum was collected and ELISA used to determine quantities ofhAAT protein within the blood. Data represents n=4 per group.

FIG. 8 shows hydrogen bonds in the VR1 region of rAAV1 and rAAV6capsids. Atom pairs involved in hydrogen bond formation were calculatedfor rAAV1 and rAAV6 crystallographic coordinates using MolProbity allatom contact analysis and visualized using KiNG graphics program.Residues comprising the VR1 loop are labeled. Hydrogen bonds generatedfrom residue 265 are visualized using green dots and are indicated byarrows. VR1 side chains are colored orange and the protein backbone iscolored yellow.

FIG. 9 shows transgene expression after injection of rAAV1 and rAAV6constructs into the eye.

FIG. 10 shows transgene expression after injection of rAAV1 and rAAV6constructs into the eye.

FIGS. 11A-11I show VR1 hydrogen bonding networks visualized by KiNGdisplay software (A). Protein main chains are depicted in orange, sidechains in light green. Hydrogen bonds are shown as dark green clouds,with arrows indicating hydrogen bonds present in VR1. VR1 residues arelabeled. In panel (B), the following bonds are highlighted for rAAV1:HG1 T265 to O S262; O A263 to HG1 T265; OG S264 to H T265. In panel (C),the following bonds are highlighted for rAAV2: OG S264 to H G265; H A266to O Q263. In panel (D), the following bonds are highlighted for rAAV3b:O Q263 to H A266; O Q263 to H G265. In panel (E), the following bondsare highlighted for rAAV4: O L258 to H N261. In panel (F), the followingbonds are highlighted for rAAV5: H S258 to O V255; O S258 to H N261. Inpanel (G), the following bonds are highlighted for rAAV6: O S264 A to HGS264 A; HG1 T265 to O S262; OG S262 to HD1 H272. In panel (H), thefollowing bonds are highlighted for rAAV8: HG1 T265 to O T265. In panel(I), the following bonds are highlighted for rAAV9: HG S265 to O S265; HG267 to O S263.

FIGS. 12A-12H show VR1 hydrogen bond networks in various rAAV serotypes.Pymol images are focused on capsid loop VR1. Dashed lines representhydrogen bonds present in the crystal structures of rAAV1 (PDB ID,3NG9), rAAV2 (PDB ID, 1LP3), rAAV3b (PDB ID, 3KIC), rAAV4 (PDB ID,2G8G), rAAV5 (PDB ID, 3NTT), rAAV6 (PDB ID, 3OAH), rAAV8 (PDB ID, 2QAO),and rAAV9 (PDB ID, 3UX1). VR1 amino acids participating in hydrogen bondnetworks are depicted as sticks and colored green. Amino acids that weremutated for this study are colored cyan. In rAAV1 (A), bonds are shownbetween residues T265 and S262, T265 and A263, T265 and S264, and S264and G266. In rAAV2 (B), bonds are shown between residues S264 and G265,and Q263 and A266. In rAAV3b (C), bonds are shown between residues Q263and A266, and Q263 and G265. In rAAV4 (D), bonds are shown betweenresidues L258 and N261. In rAAV5 (E), bonds are shown between residuesS258 and V255, and S258 and N261. In rAAV6 (F), bonds are shown betweenresidues T265 and S262, and S262 and H272. In rAAV8 (G), bonds are shownbetween residues T265 and T265 (residue hydrogen bonds to itself). InrAAV9 (H), bonds are shown between residues S263 and G267, and S265 andS265 (residue hydrogen bonds to itself).

FIGS. 13A-13F show biodistribution of rAAV1 and rAAV6 capsids bearingVR1 deletion mutations. Live-animal bioluminescent imaging of rAAV1 (A)and rAAV6 (D) capsids at 9 days post tail vein injections of 1e11 vectorgenomes per construct. At 10 dpi, indicated organs were harvested, lysedand homogenized, and luciferase expression and total protein content oflysate quantified (B, E). Values are shown in relative light units permilligram total protein (RLU/mg). An indicated subset of organs wasfurther processed via qPCR (C, F) to determine number of vector genomesper cell genomes (vg/cg). Each data point represents n=3; images are ofone representative mouse per group. Error bars reflect standarddeviation.

FIGS. 14A-14I show biodistribution of rAAV7, rAAV8, and rAAV9 capsidsbearing VR1 deletion mutations. Live-animal bioluminescent imaging ofrAAV7 (A), rAAV8 (D), and rAAV9 (G) capsids at 9 days post tail veininjections of 1e11 vector genomes per construct. At 10 dpi, indicatedorgans were harvested, lysed and homogenized, and luciferase expressionand total protein content of lysate quantified (B, E, H). Values areshown in relative light units per milligram total protein (RLU/mg). Anindicated subset of organs was further processed via qPCR (C, F, I) todetermine number of vector genomes per cell genomes (vg/cg). Each datapoint represents n=3; images are of one representative mouse per group.Error bars reflect standard deviation.

FIGS. 15A-15D show qualitative biodistribution of VR1 deletion mutantcapsids in serotypes rAAV2 and rAAV3b. In panels (A) and (B), 1e11vg ofeach of the indicated constructs was administered via the tail vein.Mice were imaged at 10 dpi. In panels (C) and (D), 5e11vg of each of theindicated constructs was administered via the tail vein. Mice wereimaged at 10 dpi. In all cases N=3 was evaluated; one representativemouse from each group is shown.

FIGS. 16A-16C show quantitative biodistribution for VR1 deletion mutantrAAV1 and rAAV6 capsids. Panels (A) and (B) depict luciferase expressionvia average RLU/mg protein for visceral organs following tail veininjection of 1e11vg of the indicated constructs. Panel (C) comparesluciferase expression via average RLU/mg protein between rAAV6/T262deland rAAV6/T265del in heart, liver and GC. N=3; error bars representstandard deviation.

FIGS. 17A-17D show quantitative and qualitative biodistribution of VR1deletion mutant rAAV7, rAAV8, and rAAV9 capsids. Panels (A), (B), and(C) depict luciferase expression via average RLU/mg protein for visceralorgans following tail vein injection of 1e11vg of the indicatedconstructs. In panel (D) mice were injected via the tail vein with1e11vg per indicated construct and imaged at 10 dpi. N=3 for allexperiments; error bars represent standard deviation. In panel (D) onerepresentative mouse from each group is shown.

FIGS. 18A-18L show biodistribution of rAAV1, rAAV2, rAAV3b, and rAAV6capsids bearing VR1 insertion/substitution mutations. Live-animalbioluminescent imaging of rAAV2 (A), rAAV3b (D), rAAV1 (G), and rAAV6(J) capsids at 9 days post tail vein injections of 1e11 vector genomesper construct. At 10 dpi, indicated organs were harvested, lysed andhomogenized, and luciferase expression and total protein content oflysate quantified (B, E, H, K). Values are shown in relative light unitsper milligram total protein (RLU/mg). An indicated subset of organs wasfurther processed via qPCR (C, F, I, L) to determine number of vectorgenomes per cell genomes (vg/cg). Each data point represents n=3; imagesare of one representative mouse per group. Error bars reflect standarddeviation.

FIGS. 19A-19D show quantitative biodistribution for VR1 265D mutantrAAV1, rAAV2, rAAV3b, and rAAV6 capsids. Panels (A), (B), (C) and (D)depict luciferase expression via average RLU/mg protein for visceralorgans following tail vein injection of 1e11vg of the indicatedconstructs. N=3; error bars represent standard deviation.

FIGS. 20A-20B show qualitative biodistribution of 265D mutant rAAV8 andrAAV9 capsids. In panels (A) and (B), 1e11vg of each of the indicatedconstructs was administered via the tail vein. Mice were imaged at 10dpi. In all cases N=3 was evaluated; one representative mouse from eachgroup is shown.

FIGS. 21A-21D show the impact of capsid heparin binding ability ontransduction efficiency of capsids bearing VR1 insertion/substationmutations. Ex vivo luciferase assay results on liver samples taken 10dpi from mice injected with 1e11vg of an rAAV2 capsid subset (A)including 265D, heparin-null, and 265D heparin null combination mutants;an rAAV3b capsid subset (B) including 265D, heparin-null, and 265Dheparin null combination mutants; and an rAAV6 capsid subset (C)including 265D, heparin-null, and 265D heparin null combination mutants.Ex vivo luciferase assay results on heart, liver, and GC samples taken10 dpi from mice injected with 1e11vg of an rAAV6 capsid subset (D)including T265del, heparin-null and T265del heparin-null combinationmutants. Data represent relative light units normalized per mg ofprotein analyzed (RLU/mg). Each data point represents n=3; error barsreflect standard deviation.

FIGS. 22A-22D show the comparison of VR1 mutant to wild-type capsidtransduction efficiency in cardiac and hepatic tissues. Ex vivoluciferase assay results on tissue samples taken from the heart (A) andliver (B) at 10 dpi of 1e11vg of a panel of VR1 deletion mutant capsids.Data represented as fold-change relative to values obtained for rAAV9,after measuring relative light units per mg of protein analyzed. Ratioof heart to liver transduction (C) of organs analyzed in A and B. Exvivo luciferase assay results on liver tissue samples (D) taken at 10dpi of 1e11vg of a panel of 265D mutant capsids. Data represented asfold-change relative to values obtained for rAAV8 after measuringrelative light units per mg of protein analyzed. Each data pointrepresents n=3; error bars reflect standard deviation.

FIGS. 23A-23B show the transduction efficiency of rAAV1 and rAAV6capsids bearing VR1 mutations in conjunction withtyrosine-to-phenylalanine mutations. Ex vivo luciferase assay results ona panel of heart, liver, and GC tissues taken from a panel of rAAV1capsids (A) bearing VR1 deletion mutations alone and in conjunction ofY445F mutations, and from a panel of rAAV6 (B) capsids with theequivalent mutations. Data represent relative light units normalized permg of protein analyzed (RLU/mg). Tissue samples were taken at 10 dpi of1e11vg per construct. Each data point represents n=3; error bars reflectstandard deviation.

FIG. 24 shows qualitative biodistribution of rAAV8 and rAAV8/S266delcapsids. 1e11vg of each of the indicated constructs was administered viathe tail vein. Mice were imaged at 10 dpi. In all cases N=3 wasevaluated; one representative mouse from each group is shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with reference to theaccompanying drawings, in which preferred embodiments of the inventionare shown. This invention may, however, be embodied in different formsand should not be construed as limited to the embodiments set forthherein. Rather, these embodiments are provided so that this disclosurewill be thorough and complete, and will fully convey the scope of theinvention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ direction, from left to right, unless specifically indicatedotherwise. Nucleotides and amino acids are represented herein in themanner recommended by the IUPAC-IUB Biochemical Nomenclature Commission,or (for amino acids) by either the one-letter code, or the three lettercode, both in accordance with 37 CFR § 1.822 and established usage. See,e.g., Patent In User Manual, 99-102 (November 1990) (U.S. Patent andTrademark Office).

Except as otherwise indicated, standard methods known to those skilledin the art may be used for the construction of recombinant parvovirusand AAV (rAAV) constructs, packaging vectors expressing the parvovirusRep and/or Cap sequences, and transiently and stably transfectedpackaging cells. Such techniques are known to those skilled in the art.See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2ndEd. (Cold Spring Harbor, N.Y., 1989); AUSUBEL et al., CURRENT PROTOCOLSIN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley &Sons, Inc., New York).

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

To illustrate further, if, for example, the specification indicates thata particular amino acid can be selected from A, G, I, L and/or V, thislanguage also indicates that the amino acid can be selected from anysubset of these amino acid(s) for example A, G, I or L; A, G, I or V; Aor G; only L; etc. as if each such subcombination is expressly set forthherein. Moreover, such language also indicates that one or more of thespecified amino acids can be disclaimed. For example, in particularembodiments the amino acid is not A, G or I; is not A; is not G or V;etc. as if each such possible disclaimer is expressly set forth herein.

Definitions

The following terms are used in the description herein and the appendedclaims.

The singular forms “a” and “an” are intended to include the plural formsas well, unless the context clearly indicates otherwise.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of the length of a polynucleotide orpolypeptide sequence, dose, time, temperature, and the like, is meant toencompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of thespecified amount.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the transitional phrase “consisting essentially of” isto be interpreted as encompassing the recited materials or steps “andthose that do not materially affect the basic and novelcharacteristic(s)” of the claimed invention (e.g., rAAV replication).See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976)(emphasis in the original); see also MPEP § 2111.03. Thus, the term“consisting essentially of” as used herein should not be interpreted asequivalent to “comprising.”

The term “consists essentially of” (and grammatical variants), asapplied to a polynucleotide or polypeptide sequence of this invention,means a polynucleotide or polypeptide that consists of both the recitedsequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5′and/or 3′ or N-terminal and/or C-terminal ends of the recited sequencesuch that the function of the polynucleotide or polypeptide is notmaterially altered. The total of ten or less additional nucleotides oramino acids includes the total number of additional nucleotides or aminoacids on both ends added together. The term “materially altered,” asapplied to polynucleotides of the invention, refers to an increase ordecrease in ability to express the encoded polypeptide of at least about50% or more as compared to the expression level of a polynucleotideconsisting of the recited sequence. The term “materially altered,” asapplied to polypeptides of the invention, refers to an increase ordecrease in transduction activity of at least about 50% or more ascompared to the activity of a polypeptide consisting of the recitedsequence.

The term “parvovirus” as used herein encompasses the familyParvoviridae, including autonomously-replicating parvoviruses anddependoviruses. The autonomous parvoviruses include members of thegenera Parvovirus, Erythrovirus, Densovirus, Iteravirus, andContravirus. Exemplary autonomous parvoviruses include, but are notlimited to, minute virus of mouse, bovine parvovirus, canine parvovirus,chicken parvovirus, feline panleukopenia virus, feline parvovirus, gooseparvovirus, H1 parvovirus, muscovy duck parvovirus, snake parvovirus,and B19 virus. Other autonomous parvoviruses are known to those skilledin the art. See, e.g., FIELDS et al., VIROLOGY, volume 2, chapter 69(4th ed., Lippincott-Raven Publishers).

The genus Dependovirus contains the adeno-associated viruses (AAV),including but not limited to, AAV type 1, AAV type 2, AAV type 3(including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAVtype 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12,AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV,equine AAV, and ovine AAV. See, e.g., FIELDS et al., VIROLOGY, volume 2,chapter 69 (4th ed., Lippincott-Raven Publishers); and Table 1.

As used herein, the term “adeno-associated virus” (AAV), includes but isnot limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3Aand 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAVtype 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV,avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV,shrimp AAV, and any other AAV now known or later discovered. See, e.g.,FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-RavenPublishers). A number of relatively new AAV serotypes and clades havebeen identified (See, e.g., Gao et al., (2004) J. Virol. 78:6381; Moriset al., (2004) Virol. 33-:375; and Table 1).

The parvovirus particles and genomes of the present invention can befrom, but are not limited to, AAV. The genomic sequences of variousserotypes of AAV and the autonomous parvoviruses, as well as thesequences of the native ITRs, Rep proteins, and capsid subunits areknown in the art. Such sequences may be found in the literature or inpublic databases such as GenBank. See, e.g., GenBank Accession NumbersNC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862,NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790,AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061,AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC_001358,NC_001540, AF513851, AF513852 and AY530579; the disclosures of which areincorporated by reference herein for teaching parvovirus and AAV nucleicacid and amino acid sequences. See also, e.g., Bantel-Schaal et al.,(1999) J. Virol. 73: 939; Chiorini et al., (1997) J. Virol. 71:6823;Chiorini et al., (1999) J. Virol. 73:1309; Gao et al., (2002) Proc. Nat.Acad. Sci. USA 99:11854; Moris et al., (2004) Virol. 33-:375-383; Moriet al., (2004) Virol. 330:375; Muramatsu et al., (1996) Virol. 221:208;Ruffing et al., (1994) J. Gen. Virol. 75:3385; Rutledge et al., (1998)J. Virol. 72:309; Schmidt et al., (2008) J. Virol. 82:8911; Shade etal., (1986) J. Virol. 58:921; Srivastava et al., (1983) J. Virol.45:555; Xiao et al., (1999) J. Virol. 73:3994; international patentpublications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Pat. No.6,156,303; the disclosures of which are incorporated by reference hereinfor teaching parvovirus and AAV nucleic acid and amino acid sequences.See also Table 1. An early description of the AAV1, AAV2 and AAV3 ITRsequences is provided by Xiao, X., (1996), “Characterization ofAdeno-associated virus (AAV) DNA replication and integration,” Ph.D.Dissertation, University of Pittsburgh, Pittsburgh, Pa. (incorporatedherein it its entirety).

TABLE 1 Complete Genomes GenBank Accession Number Adeno-associated virus1 NC_002077, AF063497 Adeno-associated virus 2 NC_001401Adeno-associated virus 3 NC_001729 Adeno-associated virus 3B NC_001863Adeno-associated virus 4 NC_001829 Adeno-associated virus 5 Y18065,AF085716 Adeno-associated virus 6 NC_001862 Avian AAV ATCC VR-865AY186198, AY629583, NC_004828 Avian AAV strain DA-1 NC_006263, AY629583Bovine AAV NC_005889, AY388617 Clade A AAV1 NC_002077, AF063497 AAV6NC_001862 Hu.48 AY530611 Hu 43 AY530606 Hu 44 AY530607 Hu 46 AY530609Clade B Hu. 19 AY530584 Hu. 20 AY530586 Hu 23 AY530589 Hu22 AY530588Hu24 AY530590 Hu21 AY530587 Hu27 AY530592 Hu28 AY530593 Hu 29 AY530594Hu63 AY530624 Hu64 AY530625 Hu13 AY530578 Hu56 AY530618 Hu57 AY530619Hu49 AY530612 Hu58 AY530620 Hu34 AY530598 Hu35 AY530599 AAV2 NC_001401Hu45 AY530608 Hu47 AY530610 Hu51 AY530613 Hu52 AY530614 Hu T41 AY695378Hu S17 AY695376 Hu T88 AY695375 Hu T71 AY695374 Hu T70 AY695373 Hu T40AY695372 Hu T32 AY695371 Hu T17 AY695370 Hu LG15 AY695377 Clade C Hu9AY530629 Hu10 AY530576 Hu11 AY530577 Hu53 AY530615 Hu55 AY530617 Hu54AY530616 Hu7 AY530628 Hu18 AY530583 Hu15 AY530580 Hu16 AY530581 Hu25AY530591 Hu60 AY530622 Ch5 AY243021 Hu3 AY530595 Hu1 AY530575 Hu4AY530602 Hu2 AY530585 Hu61 AY530623 Clade D Rh62 AY530573 Rh48 AY530561Rh54 AY530567 Rh55 AY530568 Cy2 AY243020 AAV7 AF513851 Rh35 AY243000Rh37 AY242998 Rh36 AY242999 Cy6 AY243016 Cy4 AY243018 Cy3 AY243019 Cy5AY243017 Rh13 AY243013 Clade E Rh38 AY530558 Hu66 AY530626 Hu42 AY530605Hu67 AY530627 Hu40 AY530603 Hu41 AY530604 Hu37 AY530600 Rh40 AY530559Rh2 AY243007 Bb1 AY243023 Bb2 AY243022 Rh10 AY243015 Hu17 AY530582 Hu6AY530621 Rh25 AY530557 Pi2 AY530554 Pi1 AY530553 Pi3 AY530555 Rh57AY530569 Rh50 AY530563 Rh49 AY530562 Hu39 AY530601 Rh58 AY530570 Rh61AY530572 Rh52 AY530565 Rh53 AY530566 Rh51 AY530564 Rh64 AY530574 Rh43AY530560 AAV8 AF513852 Rh8 AY242997 Rh1 AY530556 Clade F Hu14 (AAV9)AY530579 Hu31 AY530596 Hu32 AY530597 Clonal Isolate AAV5 Y18065,AF085716 AAV 3 NC_001729 AAV 3B NC_001863 AAV4 NC_001829 Rh34 AY243001Rh33 AY243002 Rh32 AY243003

The term “tropism” as used herein refers to entry of the virus into thecell, optionally and preferably followed by expression (e.g.,transcription and, optionally, translation) of sequences carried by theviral genome in the cell, e.g., for a recombinant virus, expression ofthe heterologous nucleotide sequences(s). Those skilled in the art willappreciate that transcription of a heterologous nucleic acid sequencefrom the viral genome may not be initiated in the absence oftrans-acting factors, e.g., for an inducible promoter or otherwiseregulated nucleic acid sequence. In the case of AAV, gene expressionfrom the viral genome may be from a stably integrated provirus, from anon-integrated episome, as well as any other form in which the virus maytake within the cell.

As used herein, “transduction” of a cell by parvovirus or AAV refers toparvovirus/AAV-mediated transfer of genetic material into the cell. See,e.g., FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d ed.,Lippincott-Raven Publishers).

The terms “5′ portion” and “3′ portion” are relative terms to define aspatial relationship between two or more elements. Thus, for example, a“3′ portion” of a polynucleotide indicates a segment of thepolynucleotide that is downstream of another segment. The term “3′portion” is not intended to indicate that the segment is necessarily atthe 3′ end of the polynucleotide, or even that it is necessarily in the3′ half of the polynucleotide, although it may be. Likewise, a “5′portion” of a polynucleotide indicates a segment of the polynucleotidethat is upstream of another segment. The term “5′ portion” is notintended to indicate that the segment is necessarily at the 5′ end ofthe polynucleotide, or even that it is necessarily in the 5′ half of thepolynucleotide, although it may be.

As used herein, the term “polypeptide” encompasses both peptides andproteins, unless indicated otherwise.

A “polynucleotide” is a sequence of nucleotide bases, and may be RNA,DNA or DNA-RNA hybrid sequences (including both naturally occurring andnon-naturally occurring nucleotide), and can be either single or doublestranded DNA sequences.

The term “sequence identity,” as used herein, has the standard meaningin the art. As is known in the art, a number of different programs canbe used to identify whether a polynucleotide or polypeptide has sequenceidentity or similarity to a known sequence. Sequence identity orsimilarity may be determined using standard techniques known in the art,including, but not limited to, the local sequence identity algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequenceidentity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12:387 (1984), preferably using the defaultsettings, or by inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351 (1987); the method is similar to that described by Higgins &Sharp, CABIOS 5:151 (1989).

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215:403 (1990) and Karlin et al.,Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., Meth. Enzymol., 266:460 (1996); blast.wustl/edu/blast/README.html.WU-BLAST-2 uses several search parameters, which are preferably set tothe default values. The parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched; however, the valuesmay be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., Nucleic Acids Res. 25:3389 (1997).

A percentage amino acid sequence identity value is determined by thenumber of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

In a similar manner, percent nucleic acid sequence identity is definedas the percentage of nucleotide residues in the candidate sequence thatare identical with the nucleotides in the polynucleotide specificallydisclosed herein.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer nucleotides than the polynucleotides specifically disclosedherein, it is understood that in one embodiment, the percentage ofsequence identity will be determined based on the number of identicalnucleotides in relation to the total number of nucleotides. Thus, forexample, sequence identity of sequences shorter than a sequencespecifically disclosed herein, will be determined using the number ofnucleotides in the shorter sequence, in one embodiment. In percentidentity calculations relative weight is not assigned to variousmanifestations of sequence variation, such as insertions, deletions,substitutions, etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0,”which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

As used herein, an “isolated” polynucleotide (e.g., an “isolated DNA” oran “isolated RNA”) means a polynucleotide separated or substantiallyfree from at least some of the other components of the naturallyoccurring organism or virus, for example, the cell or viral structuralcomponents or other polypeptides or nucleic acids commonly foundassociated with the polynucleotide.

Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide.

A “therapeutic polypeptide” is a polypeptide that may alleviate orreduce symptoms that result from an absence or defect in a protein in acell or subject. Alternatively, a “therapeutic polypeptide” is one thatotherwise confers a benefit to a subject, e.g., anti-cancer effects orimprovement in transplant survivability.

As used herein, the term “modified,” as applied to a polynucleotide orpolypeptide sequence, refers to a sequence that differs from a wild-typesequence due to one or more deletions, additions, substitutions, or anycombination thereof.

As used herein, by “isolate” or “purify” (or grammatical equivalents) avirus vector, it is meant that the virus vector is at least partiallyseparated from at least some of the other components in the startingmaterial.

By the terms “treat,” “treating,” or “treatment of” (and grammaticalvariations thereof) it is meant that the severity of the subject'scondition is reduced, at least partially improved or stabilized and/orthat some alleviation, mitigation, decrease or stabilization in at leastone clinical symptom is achieved and/or there is a delay in theprogression of the disease or disorder.

The terms “prevent,” “preventing,” and “prevention” (and grammaticalvariations thereof) refer to prevention and/or delay of the onset of adisease, disorder and/or a clinical symptom(s) in a subject and/or areduction in the severity of the onset of the disease, disorder and/orclinical symptom(s) relative to what would occur in the absence of themethods of the invention. The prevention can be complete, e.g., thetotal absence of the disease, disorder and/or clinical symptom(s). Theprevention can also be partial, such that the occurrence of the disease,disorder and/or clinical symptom(s) in the subject and/or the severityof onset is less than what would occur in the absence of the presentinvention.

A “treatment effective” amount as used herein is an amount that issufficient to provide some improvement or benefit to the subject.Alternatively stated, a “treatment effective” amount is an amount thatwill provide some alleviation, mitigation, decrease or stabilization inat least one clinical symptom in the subject. Those skilled in the artwill appreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

A “prevention effective” amount as used herein is an amount that issufficient to prevent and/or delay the onset of a disease, disorderand/or clinical symptoms in a subject and/or to reduce and/or delay theseverity of the onset of a disease, disorder and/or clinical symptoms ina subject relative to what would occur in the absence of the methods ofthe invention. Those skilled in the art will appreciate that the levelof prevention need not be complete, as long as some benefit is providedto the subject.

The terms “heterologous nucleotide sequence” and “heterologous nucleicacid” are used interchangeably herein and refer to a sequence that isnot naturally occurring in the virus. In some embodiments, theheterologous nucleic acid comprises an open reading frame that encodes apolypeptide or nontranslated RNA of interest (e.g., for delivery to acell or subject).

As used herein, the terms “virus vector,” “vector” or “gene deliveryvector” refer to a virus (e.g., AAV) particle that functions as anucleic acid delivery vehicle, and which comprises the vector genome(e.g., viral DNA [vDNA]) packaged within a virion. Alternatively, insome contexts, the term “vector” may be used to refer to the vectorgenome/vDNA alone or a plasmid.

The virus vectors of the invention can further be duplexed parvovirusparticles as described in international patent publication WO 01/92551(the disclosure of which is incorporated herein by reference in itsentirety). Thus, in some embodiments, double stranded (duplex) genomescan be packaged.

A “rAAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA)that comprises one or more heterologous nucleic acid sequences. rAAVvectors generally require only the 145 base ITR in cis to generatevirus. All other viral sequences are dispensable and may be supplied intrans (Muzyczka (1992) Curr. Topics Microbiol. Immunol. 158:97).Typically, the rAAV vector genome will only retain the one or more ITRsequence so as to maximize the size of the transgene that can beefficiently packaged by the vector. The structural and non-structuralprotein coding sequences may be provided in trans (e.g., from a vector,such as a plasmid, or by stably integrating the sequences into apackaging cell). In embodiments of the invention the rAAV vector genomecomprises at least one ITR sequence (e.g., AAV ITR sequence), optionallytwo ITRs (e.g., two AAV ITRs), which typically will be at the 5′ and 3′ends of the vector genome and flank the heterologous nucleic acid, butneed not be contiguous thereto. The ITRs can be the same or differentfrom each other.

The term “terminal repeat” or “TR” includes any viral terminal repeat orsynthetic sequence that forms a hairpin structure and functions as aninverted terminal repeat (i.e., mediates the desired functions such asreplication, virus packaging, integration and/or provirus rescue, andthe like). The ITR can be an AAV ITR or a non-AAV ITR. For example, anon-AAV ITR sequence such as those of other parvoviruses (e.g., canineparvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus,human parvovirus B-19) or the SV40 hairpin that serves as the origin ofSV40 replication can be used as an ITR, which can further be modified bytruncation, substitution, deletion, insertion and/or addition. Further,the ITR can be partially or completely synthetic, such as the “double-Dsequence” as described in U.S. Pat. No. 5,478,745 to Samulski et al.FIG. 24 provides examples of synthetic ITRs contemplated by the presentinvention.

Parvovirus genomes have palindromic sequences at both their 5′ and 3′ends. The palindromic nature of the sequences leads to the formation ofa hairpin structure that is stabilized by the formation of hydrogenbonds between the complementary base pairs. This hairpin structure isbelieved to adopt a “Y” or a “T” shape. See, e.g., FIELDS et al.,VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-RavenPublishers).

An “AAV inverted terminal repeat” or “AAV ITR” may be from any AAV,including but not limited to serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9,10, 11, or 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV,ovine AAV, goat AAV, shrimp AAV, or any other AAV now known or laterdiscovered (see, e.g., Table 1). An AAV ITR need not have the nativeterminal repeat sequence (e.g., a native AAV ITR sequence may be alteredby insertion, deletion, truncation and/or missense mutations), as longas the terminal repeat mediates the desired functions, e.g.,replication, virus packaging, persistence, and/or provirus rescue, andthe like.

The virus vectors of the invention can further be “targeted” virusvectors (e.g., having a directed tropism) and/or a “hybrid” parvovirus(i.e., in which the viral ITRs and viral capsid are from differentparvoviruses) as described in international patent publication WO00/28004 and Chao et al., (2000) Mol. Therapy 2:619.

Further, the viral capsid or genomic elements can contain othermodifications, including insertions, deletions and/or substitutions.

As used herein, the term “amino acid” encompasses any naturallyoccurring amino acids, modified forms thereof, and synthetic aminoacids.

Naturally occurring, levorotatory (L-) amino acids are shown in Table 2.

TABLE 2 Abbreviation Amino Acid Residue Three-Letter Code One-LetterCode Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid(Aspartate) Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid(Glutamate) Glu E Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

Alternatively, the amino acid can be a modified amino acid residue(nonlimiting examples are shown in Table 3) or can be an amino acid thatis modified by post-translation modification (e.g., acetylation,amidation, formylation, hydroxylation, methylation, phosphorylation orsulfatation).

TABLE 3 Amino Acid Residue Derivatives Modified Amino Acid ResidueAbbreviation 2-Aminoadipic acid Aad 3-Aminoadipic acid bAadbeta-Alanine, beta-Aminoproprionic acid bAla 2-Aminobutyric acid Abu4-Aminobutyric acid, Piperidinic acid 4Abu 6-Aminocaproic acid Acp2-Aminoheptanoic acid Ahe 2-Aminoisobutyric acid Aib 3-Aminoisobutyricacid bAib 2-Aminopimelic acid Apm t-butylalanine t-BuA Citrulline CitCyclohexylalanine Cha 2,4-Diaminobutyric acid Dbu Desmosine Des2,2′-Diaminopimelic acid Dpm 2,3-Diaminoproprionic acid DprN-Ethylglycine EtGly N-Ethylasparagine EtAsn Homoarginine hArgHomocysteine hCys Homoserine hSer Hydroxylysine Hyl Allo-HydroxylysineaHyl 3-Hydroxyproline 3Hyp 4-Hydroxyproline 4Hyp Isodesmosine Ideallo-Isoleucine aIle Methionine sulfoxide MSO N-Methylglycine, sarcosineMeGly N-Methylisoleucine MeIle 6-N-Methyllysine MeLys N-MethylvalineMeVal 2-Naphthylalanine 2-Nal Norvaline Nva Norleucine Nle Ornithine Orn4-Chlorophenylalanine Phe(4-Cl) 2-Fluorophenylalanine Phe(2-F)3-Fluorophenylalanine Phe(3-F) 4-Fluorophenylalanine Phe(4-F)Phenylglycine Phg Beta-2-thienylalanine Thi

Further, the non-naturally occurring amino acid can be an “unnatural”amino acid as described by Wang et al., (2006) Annu. Rev. Biophys.Biomol. Struct. 35:225-49. These unnatural amino acids canadvantageously be used to chemically link molecules of interest to theAAV capsid protein.

The term “template” or “substrate” is used herein to refer to apolynucleotide sequence that may be replicated to produce the parvovirusviral DNA. For the purpose of vector production, the template willtypically be embedded within a larger nucleotide sequence or construct,including but not limited to a plasmid, naked DNA vector, bacterialartificial chromosome (BAC), yeast artificial chromosome (YAC) or aviral vector (e.g., adenovirus, herpesvirus, Epstein-Barr Virus, AAV,baculoviral, retroviral vectors, and the like). Alternatively, thetemplate may be stably incorporated into the chromosome of a packagingcell.

As used herein, parvovirus or AAV “Rep coding sequences” indicate thenucleic acid sequences that encode the parvoviral or AAV non-structuralproteins that mediate viral replication and the production of new virusparticles. The parvovirus and AAV replication genes and proteins havebeen described in, e.g., FIELDS et al., VIROLOGY, volume 2, chapters 69& 70 (4th ed., Lippincott-Raven Publishers).

The “Rep coding sequences” need not encode all of the parvoviral or AAVRep proteins. For example, with respect to AAV, the Rep coding sequencesdo not need to encode all four AAV Rep proteins (Rep78, Rep 68, Rep52and Rep40), in fact, it is believed that AAV5 only expresses the splicedRep68 and Rep40 proteins. In representative embodiments, the Rep codingsequences encode at least those replication proteins that are necessaryfor viral genome replication and packaging into new virions. The Repcoding sequences will generally encode at least one large Rep protein(i.e., Rep78/68) and one small Rep protein (i.e., Rep52/40). Inparticular embodiments, the Rep coding sequences encode the AAV Rep78protein and the AAV Rep52 and/or Rep40 proteins. In other embodiments,the Rep coding sequences encode the Rep68 and the Rep52 and/or Rep40proteins. In a still further embodiment, the Rep coding sequences encodethe Rep68 and Rep52 proteins, Rep68 and Rep40 proteins, Rep78 and Rep52proteins, or Rep78 and Rep40 proteins.

As used herein, the term “large Rep protein” refers to Rep68 and/orRep78. Large Rep proteins of the claimed invention may be eitherwild-type or synthetic. A wild-type large Rep protein may be from anyparvovirus or AAV, including but not limited to serotypes 1, 2, 3a, 3b,4, 5, 6, 7, 8, 9, 10, 11, or 13, or any other AAV now known or laterdiscovered (see, e.g., Table 1). A synthetic large Rep protein may bealtered by insertion, deletion, truncation and/or missense mutations.

Those skilled in the art will further appreciate that it is notnecessary that the replication proteins be encoded by the samepolynucleotide. For example, for MVM, the NS-1 and NS-2 proteins (whichare splice variants) may be expressed independently of one another.Likewise, for AAV, the p19 promoter may be inactivated and the large Repprotein(s) expressed from one polynucleotide and the small Repprotein(s) expressed from a different polynucleotide. Typically,however, it will be more convenient to express the replication proteinsfrom a single construct. In some systems, the viral promoters (e.g., AAVp19 promoter) may not be recognized by the cell, and it is thereforenecessary to express the large and small Rep proteins from separateexpression cassettes. In other instances, it may be desirable to expressthe large Rep and small Rep proteins separately, i.e., under the controlof separate transcriptional and/or translational control elements. Forexample, it may be desirable to control expression of the large Repproteins, so as to decrease the ratio of large to small Rep proteins. Inthe case of insect cells, it may be advantageous to down-regulateexpression of the large Rep proteins (e.g., Rep78/68) to avoid toxicityto the cells (see, e.g., Urabe et al., (2002) Human Gene Therapy13:1935).

As used herein, the parvovirus or AAV “cap coding sequences” encode thestructural proteins that form a functional parvovirus or AAV capsid(i.e., can package DNA and infect target cells). Typically, the capcoding sequences will encode all of the parvovirus or AAV capsidsubunits, but less than all of the capsid subunits may be encoded aslong as a functional capsid is produced. Typically, but not necessarily,the cap coding sequences will be present on a single nucleic acidmolecule.

The capsid structure of autonomous parvoviruses and AAV are described inmore detail in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69& 70 (4th ed., Lippincott-Raven Publishers).

Modified Parvovirus Capsid Proteins

The present invention provides modified parvovirus capsid proteins thatprovide enhanced tissue transduction capabilities and/or modified tissuespecificities and can be used to prepare parvovirus vectors forefficient delivery of nucleic acids to cells. The inventors havediscovered that destabilization of the variable region 1 (VR1) loop ofthe capsid protein by disruption of hydrogen bonding between amino acidresidues results in enhanced cell transduction and/or modified tissuespecificities.

One aspect of the invention relates to a parvovirus capsid proteincomprising a capsid protein amino acid sequence from an AAV serotype orany other parvovirus with an icosahedral capsid structure of T=1,wherein the variable region 1 (VR1) loop comprising amino acid residues258 to 272 of AAV1 capsid protein or the corresponding amino acidresidues from another AAV or parvovirus capsid protein is modified bydeletion and/or substitution of one or more amino acid residues to causeregional destabilization within the loop due to the targeted destructionof hydrogen bonding patterns orchestrated by the residues, wherein thecapsid protein comprising the modification provides to a virus vectorcomprising the capsid protein increased transduction efficiency and/ormodified tissue specificity relative to a virus vector comprising acapsid protein that does not contain the modification.

AAV serotypes are well known in the art and are described above inTable 1. AAV serotypes have an icosahedral capsid structure of T=1.

There are 8 genera under the subfamily Parvoviridae that are capable ofinfecting vertebrates, and all are distinguished by capsids bearing T=1icosahedral symmetry. Parvoviridae include, without limitation, thefollowing viruses.

1.) Amdoparvovirus (species include: Aleutian mink disease virus, Grayfox amdovirus)

2.) Aveparvovirus (species include: Aveparvovirus)

3.) Bocaparvovirus (species include: Carnivore bocaparvovirus 1-3;Pinniped bocaparvovirus 1 and 2; Primate bocaparvovirus 1 and 2;Ungulate bocaparvovirus 1-5)

4.) Copiparvovirus (species include: Copiparvovirus)

5.) Dependoparvovirus (species include: Adeno-associateddependoparvovirus A; Adeno-associated dependoparvovirus B; Anseriformdependoparvovirus 1; Avian dependoparvovirus 1; Chiropterandependoparvovirus 1; Pinniped dependoparvovirus 1; Squamatedependoparvovirus 1)6.) Erythroparvovirus (species include: Primate erythroparvovirus 1-4;Rodent erythroparvovirus 1; Ungulate erythroparvovirus 1)7.) Protoparvovirus (Species include: Carnivore protoparvovirus 1;Rodent protoparvovirus 1 and 2; Ungulate parvovirus 1)8.) Tetraparvovirus (Species include: Chiropteran tetraparvovirus 1;Primate tetraparvovirus 1; Ungulate tetraparvovirus 1-4)

Capsid protein sequences are known in the art and are available insequence databases such as GenBank. The amino acid residue numbers usedherein with respect to the capsid protein from AAV1, AAV2, AAV3b, AAV4,AAV5, AAV6, AAV7, AAV8, and AAV9 refer to the sequences as disclosed inGenBank Accession Nos. as listed in Table 1.

The VR1 region is an art-recognized portion of the capsid proteinsequence of AAV serotypes and parvoviruses with an icosahedral capsidstructure of T=1. The loop that makes up the VR1 region consistsessentially of amino acid residues 258 to 272 of AAV1 or thecorresponding amino acid residues from another AAV or parvovirus.Because the VR1 loop is a well recognized structure, the correspondingamino acid residues may be readily identified by one of skill in theart. For example, Table 4 lists includes a list of exemplary VR1 aminoacid residues. While specific residues are listed, it will be understoodby one of skill in the art that the boundaries of the VR1 region areapproximate and may vary by one or two residues.

TABLE 4 Parvovirus VR1 Amino Acid Residues AAV1 ₂₆₁SSASTGASNDNHY₂₇₃(SEQ ID NO: 1) rAAV2 ₂₆₁SSQSGAS₂₆₇ (SEQ ID NO: 2) rAAV3b₂₆₁SSQSGASNDNHY₂₇₂ (SEQ ID NO: 3) rAAV4 ₂₅₆ESLQSNTY₂₆₃ (SEQ ID NO: 4)rAAV5 ₂₅₂SGSVDGS₂₅₈ (SEQ ID NO: 5) rAAV6 ₂₆₁SSASTGASNDNHY₂₇₃(SEQ ID NO: 6) rAAV8 ₂₆₃NGTSGGAT₂₇₀ (SEQ ID NO: 7) rAAV9 ₂₆₁SNSTSGGSS₂₆₉(SEQ ID NO: 8)

The VR1 region of parvovirus and AAV is characterized by a number ofhydrogen bonds that stabilize the region. For example, the AAV1 capsidcontains four hydrogen bonds in the VR1 region and the AAV6 capsidcontains two hydrogen bonds in the BR1 region (see FIG. 8). Thestability of the VR1 loop can be reduced by eliminating one or more ofthe hydrogen bonds in the region, e.g., by deleting an amino acidresidue involved in hydrogen bonding or substituting the amino acidresidue with a residue that does not form a hydrogen bond. In someembodiments, at least 1, 2, 3, or 4 or more hydrogen bonds are disruptedwithin the VR1 loop.

Hydrogen bonds formed by amino acid residues in the VR1 loop can beidentified by methods known in the art, including the computationalanalysis of capsid protein crystal structures, capsid protein nuclearmagnetic resonance structures, comparisons of sequence homology, etc.Examples of amino acid residues directly involved in hydrogen bonding inthe VR1 loop are listed in Table 5.

TABLE 5 Virus Hydrogen Bonds AAV1 HG1 T265 to O S262 O A263 to HG1 T265OG S264 to H T265 AAV2 OG S264 to H G265 H A266 to O Q263 AAV3b O Q263to H A266 O Q263 to H G265 AAV4 O L258 to H N261 AAV5 H S258 to O V255 OS258 to H N261 AAV6 O S264 A to HG S264 A HG1 T265 to O S262 OG S262 toHD1 H272 AAV8 HG1 T265 to O T265 AAV9 HG S265 to O S265 H G267 to O S263

For example, crystal structures may be downloaded from public databases,e.g., the RCSB Protein Data Bank (rcsb.org), a publicly accessiblerepository for all structures that were generated using NIH funding.Individual pdb files may be run through a structural validity program,such as the publicly available MolProbity4 structural validation program(molprobidity.biochem.duke.edu), which analyzes the geometricrelationships between individual molecules within a crystal structure aswell as their electron-cloud positions in order to provide visualinformation on the location of hydrogen bonding in a given structure/pdbfile. The output of MolProbity is a Kinemage, which is a graphicrepresentation of the crystal structure with included hydrogen bonding,and which can be visualized using the publicly available softwareprogram KING (kinemage.biochem.duke.edu).

In some embodiments, the transduction efficiency of a virus vectorcomprising the modified capsid protein is increased at least about 10%relative to a virus vector comprising a capsid protein that does notcontain the modification, e.g., at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or more. The capsid proteinthat does not contain the modification may be a wild-type capsid proteinor may be a synthetic capsid protein as long as it does not contain themodifications of the present invention. Transduction efficiency may bemeasured by techniques well known in the art and as described herein.

In some embodiments, the tissue specificity of a virus vector comprisingthe modified capsid protein is altered. In certain embodiments, thetransduction of muscle, e.g., skeletal and/or cardiac muscle, isincreased at least about 10% relative to a virus vector comprising acapsid protein that does not contain the modification, e.g., at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,500%, or more. In certain embodiments, the transduction of liver isdecreased at least about 10% relative to a virus vector comprising acapsid protein that does not contain the modification, e.g., at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,500%, or more. In some embodiments, the transduction of muscle isincreased and the transduction of liver is decreased relative to a virusvector comprising a capsid protein that does not contain themodification. In certain embodiments, the transduction of liver isincreased at least about 10% relative to a virus vector comprising acapsid protein that does not contain the modification, e.g., at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,500%, or more.

In some embodiments, the capsid is modified by deleting or substitutingone or more amino acid residues of amino acid residues 258 to 272 ofAAV1 capsid protein or the corresponding amino acid residues fromanother AAV or parvovirus capsid protein, e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more residues are deleted or substituted. The deleted orsubstituted residue may be any residue in the VR1 that results in achange in rotamer state for residues that are participating in hydrogenbond networks. In another embodiment, the capsid is modified by deletingor substituting one or more amino acid residues of amino acid residues261 to 269 of AAV1 capsid protein or the corresponding amino acidresidues from another AAV or parvovirus capsid protein. In a furtherembodiment, the capsid is modified by deleting or substituting aminoacid residue 265 of AAV1 capsid protein or the corresponding amino acidresidues from another AAV or parvovirus capsid protein.

In certain embodiments, the modification is made to a AAV1 or AAV6capsid, resulting in an increase in muscle transduction and a decreasein liver transduction. In certain embodiments, the modification is madeto a AAV7, AAV8, or AAV9 capsid, resulting in and a decrease in livertransduction while maintaining muscle transduction. In some embodiments,the modification is one or more of the modifications shown in Table 6.In certain embodiments, the modified capsid is not from AAV2, AAV3b,AAV4, or AAV5.

TABLE 6 Variable region 1 deletion mutations. Serotype Mutations rAAV1T265del rAAV2 Q263del, S264del rAAV3b Q263del rAAV4 N261del rAAV5S258del rAAV6 S262del, T265del rAAV7 T265del rAAV8 T265del rAAV9S263del, S265del, S263del_S265del

In some embodiments, the capsid is modified by inserting one or moreamino acid residues (e.g., an aspartic acid) after amino acid residue264 of AAV2 capsid protein or the corresponding amino acid residues fromanother AAV or parvovirus capsid protein. In another embodiment, thecapsid is modified by substituting an amino acid residue of amino acidresidue 265 (e.g., with an aspartic acid) of AAV1 capsid protein or thecorresponding amino acid residues from another AAV or parvovirus capsidprotein.

In certain embodiments, the modification is made to a AAV1, AAV2, orAAV3b capsid, resulting in an increase in liver transduction. In certainembodiments, the modification is made to a AAV6 capsid, resulting in anincrease in liver transduction and muscle transduction. In certainembodiments, the transduction of liver is increased at least about 10%relative to a virus vector comprising a capsid protein that does notcontain the modification, e.g., at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or more. In otherembodiments, the modification is one or more of the modifications shownin Table 7.

TABLE 7 Variable region 1 aspartic acid insertion and substitutionmutations. Serotype Mutations rAAV1 T265D rAAV2 265D rAAV3b 266D rAAV6T265D rAAV8 T265D rAAV9 S265D

A further aspect of the invention relates to the effect of reducing theheparin sulfate binding capability of capsid proteins on transductionefficiency. The examples herein demonstrate that substantially reducingthe binding of the capsid protein to heparin sulfate enhancestransduction efficiency. It is further demonstrated that, in someinstances, the substantial reduction of heparin sulfate bindingcapability allosterically interacts with destabilization of the VR1 loopto provide a substantial increase in transduction efficiency compared toeither modification alone.

In one aspect of the invention, the capsid protein comprising amodification in the VR1 loop is from an AAV serotype or other parvovirusthat binds to heparin sulfate, wherein one or more amino acid residuesthat mediate binding of the capsid protein to heparin sulfate aresubstituted and/or deleted, wherein binding of the capsid protein toheparin sulfate is substantially reduced. The term “substantiallyreduced,” as used herein, refers to a reduction in heparin sulfatebinding ability of at least about 80%, e.g., at least about 85%, 90%,95%, 96%, 97%, 98%, or 99%, or more. The binding of capsid proteins toheparin sulfate can be measured by techniques well known in the art andas described herein.

In some embodiments, the capsid protein that binds heparin sulfate isfrom an AAV2, AAV3a, AAV3b, AAV6, or AAV8 serotype. In some embodiments,the capsid protein that binds heparin sulfate is from an AAV3a, AAV3b,AAV6, or AAV8 serotype. The amino acid residue(s) involved in heparinsulfate binding in each of these serotypes are well known in the art.Examples include, without limitation, R484, R487, K532, R585, and R588in AAV2, K531 in AAV6, and R594 in AAV3b. In some embodiments, thecapsid protein that binds heparin sulfate includes any synthetic AAV orparvovirus capsid protein that has been modified to provide heparinsulfate binding capability.

In one embodiment, the capsid protein comprises the amino acid sequencefrom an AAV6 serotype, wherein amino acid residue 531 has beensubstituted to substantially reduce binding to heparin sulfate, e.g., bysubstitution with glutamic acid. In another embodiment, the capsidprotein comprises the amino acid sequence from an AAV2 serotype, whereinamino acid residue 585 has been substituted to substantially reducebinding to heparin sulfate, e.g., by substitution with glutamic acid. Inanother embodiment, the capsid protein comprises the amino acid sequencefrom an AAV3b serotype, wherein amino acid residue 594 has beensubstituted to substantially reduce binding to heparin sulfate, e.g., bysubstitution with alanine.

Another aspect of the invention relates to capsid proteins in which oneor more amino acid residues that mediate binding of the capsid proteinto heparin sulfate are substituted and/or deleted, wherein binding ofthe capsid protein to heparin sulfate is substantially reduced. In someembodiments, these capsid proteins do not have a modification in the VR1loop. In some embodiments, the capsid protein comprises an amino acidsequence from an AAV3a, AAV3b, AAV6, or AAV8 serotype. In oneembodiment, the capsid protein comprises the amino acid sequence from anAAV6 serotype, wherein amino acid residue 531 has been substituted tosubstantially reduce binding to heparin sulfate, e.g., by substitutionwith glutamic acid.

A further aspect of the invention relates to an AAV capsid proteincomprising an amino acid sequence from an AAV2, AAV3a, or AAV3bserotype, wherein the capsid protein comprises an insertion of one ormore amino acid residues immediately following residue 264 of AAV2capsid protein or the corresponding residue of AAV3a or AAV3b capsidprotein, and one or more amino acid residues that mediate binding of thecapsid protein to heparin sulfate are substituted and/or deleted,wherein binding of the capsid protein to heparin sulfate issubstantially reduced; wherein the capsid protein provides to a virusvector comprising the capsid protein increased transduction efficiencyrelative to a virus vector comprising an unmodified capsid protein.

The insertion of one or more amino acid residues immediately followingresidue 264 of AAV2 capsid protein has been demonstrated to enhancedtransduction efficiency (see, e.g., U.S. Pat. No. 7,892,809,incorporated herein by reference in its entirety). In one embodiment,the insertion immediately following residue 264 of AAV2 capsid proteinor the corresponding residue of AAV3a or AAV3b capsid protein is of anaspartic acid, glutamic acid, or phenylalanine residue. In oneembodiment, the capsid protein comprises an amino acid sequence from anAAV2 serotype, wherein amino acid residue 585 has been substituted tosubstantially reduce binding to heparin sulfate, e.g., with glutamicacid.

A further aspect of the invention relates to an AAV capsid proteincomprising a deletion and/or substitution of one or more amino acidresidues of amino acid residues 258 to 272 of AAV1 capsid protein or thecorresponding amino acid residues from another AAV or parvovirus capsidprotein and further comprising a substitution of a tyrosine residue,e.g., to improve cardiac transduction efficiency. Mutation of selecttyrosine residues on the rAAV capsid surface has been shown to enhancerAAV transduction efficiency by avoiding the phosphorylation andultimate proteasomal degradation of intracellular capsids before theyare able to reach the nucleus (Zhong et al., Virology 381(2):194(2008)). In some embodiments the tyrosine substitution is Y445F in AAV1or AAV6.

One aspect of the invention relates to a polynucleotide encoding thecapsid protein of the invention. In some embodiments, the polynucleotidecomprises, consists essentially of, or consists of a nucleotide sequenceencoding the capsid protein of the invention, e.g., one of the sequencesdisclosed herein. In other embodiments, the polynucleotide comprises,consists essentially of, or consists of a nucleotide sequence encoding acapsid protein that is at least 80% identical, e.g., at least 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to one of the capsid proteinsequences disclosed herein.

An additional other aspect of the invention relates to a parvoviruscapsid comprising the capsid protein of the invention. In someembodiments, all of the capsid proteins in the capsid are capsidproteins of the invention. In other embodiments, some but not all of thecapsid proteins in the capsid are capsid proteins of the invention.

The invention also provides a viral vector comprising the parvoviruscapsid of the invention. The viral vector may be a parvovirus vector,e.g., an AAV vector. The viral vector may further comprise a nucleicacid comprising a recombinant viral template, wherein the nucleic acidis encapsidated by the parvovirus capsid. The invention further providesa recombinant parvovirus particle (e.g., a recombinant AAV particle)comprising the capsid protein of the invention. Viral vectors and viralparticles are discussed further below.

In certain embodiments, the viral vector exhibits a modified tropism dueto the presence of the capsid protein of the invention. In oneembodiment, the parvovirus vector exhibits systemic tropism forskeletal, cardiac muscle, and/or diaphragm muscle, e.g., for skeletalmuscle. In other embodiments, the parvovirus vector has reduced tropismfor liver compared to a virus vector comprising a wild-type capsidprotein.

Methods of Producing Virus Vectors

The present invention further provides methods of producing virusvectors. In one particular embodiment, the present invention provides amethod of producing a recombinant parvovirus particle, comprisingproviding to a cell permissive for parvovirus replication: (a) arecombinant parvovirus template comprising (i) a heterologous nucleotidesequence, and (ii) a parvovirus ITR; (b) a polynucleotide comprising Repcoding sequences and the Cap coding sequences of the invention; underconditions sufficient for the replication and packaging of therecombinant parvovirus template; whereby recombinant parvovirusparticles are produced in the cell. Conditions sufficient for thereplication and packaging of the recombinant parvovirus template can be,e.g., the presence of AAV sequences sufficient for replication of theparvovirus template and encapsidation into parvovirus capsids (e.g.,parvovirus rep sequences and parvovirus cap sequences) and helpersequences from adenovirus and/or herpesvirus. In particular embodiments,the parvovirus template comprises two parvovirus ITR sequences, whichare located 5′ and 3′ to the heterologous nucleic acid sequence,although they need not be directly contiguous thereto.

In some embodiments, the recombinant parvovirus template comprises anITR that is not resolved by Rep to make duplexed AAV vectors asdescribed in international patent publication WO 01/92551.

The parvovirus template and parvovirus rep and cap sequences areprovided under conditions such that virus vector comprising theparvovirus template packaged within the parvovirus capsid is produced inthe cell. The method can further comprise the step of collecting thevirus vector from the cell. The virus vector can be collected from themedium and/or by lysing the cells.

The cell can be a cell that is permissive for parvoviral viralreplication. Any suitable cell known in the art may be employed. Inparticular embodiments, the cell is a mammalian cell (e.g., a primate orhuman cell). As another option, the cell can be a trans-complementingpackaging cell line that provides functions deleted from areplication-defective helper virus, e.g., 293 cells or other E1atrans-complementing cells.

The parvovirus replication and capsid sequences may be provided by anymethod known in the art. Current protocols typically express theparvovirus rep/cap genes on a single plasmid. The parvovirus replicationand packaging sequences need not be provided together, although it maybe convenient to do so. The parvovirus rep and/or cap sequences may beprovided by any viral or non-viral vector. For example, the rep/capsequences may be provided by a hybrid adenovirus or herpesvirus vector(e.g., inserted into the E1a or E3 regions of a deleted adenovirusvector). EBV vectors may also be employed to express the parvovirus capand rep genes. One advantage of this method is that EBV vectors areepisomal, yet will maintain a high copy number throughout successivecell divisions (i.e., are stably integrated into the cell asextra-chromosomal elements, designated as an “EBV based nuclearepisome,” see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).

As a further alternative, the rep/cap sequences may be stablyincorporated into a cell.

Typically the parvovirus rep/cap sequences will not be flanked by theTRs, to prevent rescue and/or packaging of these sequences.

The parvovirus template can be provided to the cell using any methodknown in the art. For example, the template can be supplied by anon-viral (e.g., plasmid) or viral vector. In particular embodiments,the parvovirus template is supplied by a herpesvirus or adenovirusvector (e.g., inserted into the E1a or E3 regions of a deletedadenovirus). As another illustration, Palombo et al., (1998) J. Virology72:5025, describes a baculovirus vector carrying a reporter gene flankedby the AAV TRs. EBV vectors may also be employed to deliver thetemplate, as described above with respect to the rep/cap genes.

In another representative embodiment, the parvovirus template isprovided by a replicating rAAV virus. In still other embodiments, an AAVprovirus comprising the parvovirus template is stably integrated intothe chromosome of the cell.

To enhance virus titers, helper virus functions (e.g., adenovirus orherpesvirus) that promote a productive parvovirus infection can beprovided to the cell. Helper virus sequences necessary for parvovirusreplication are known in the art. Typically, these sequences will beprovided by a helper adenovirus or herpesvirus vector. Alternatively,the adenovirus or herpesvirus sequences can be provided by anothernon-viral or viral vector, e.g., as a non-infectious adenovirusminiplasmid that carries all of the helper genes that promote efficientparvovirus production as described by Ferrari et al., (1997) Nature Med.3:1295, and U.S. Pat. Nos. 6,040,183 and 6,093,570.

Further, the helper virus functions may be provided by a packaging cellwith the helper sequences embedded in the chromosome or maintained as astable extrachromosomal element. Generally, the helper virus sequencescannot be packaged into AAV virions, e.g., are not flanked by ITRs.

Those skilled in the art will appreciate that it may be advantageous toprovide the parvovirus replication and capsid sequences and the helpervirus sequences (e.g., adenovirus sequences) on a single helperconstruct. This helper construct may be a non-viral or viral construct.As one nonlimiting illustration, the helper construct can be a hybridadenovirus or hybrid herpesvirus comprising the AAV rep/cap genes.

In one particular embodiment, the parvovirus rep/cap sequences and theadenovirus helper sequences are supplied by a single adenovirus helpervector. This vector can further comprise the parvovirus template. Theparvovirus rep/cap sequences and/or the parvovirus template can beinserted into a deleted region (e.g., the E1a or E3 regions) of theadenovirus.

In a further embodiment, the parvovirus rep/cap sequences and theadenovirus helper sequences are supplied by a single adenovirus helpervector. According to this embodiment, the parvovirus template can beprovided as a plasmid template.

In another illustrative embodiment, the parvovirus rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helpervector, and the parvovirus template is integrated into the cell as aprovirus. Alternatively, the parvovirus template is provided by an EBVvector that is maintained within the cell as an extrachromosomal element(e.g., as an EBV based nuclear episome).

In a further exemplary embodiment, the parvovirus rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helper.The parvovirus template can be provided as a separate replicating viralvector. For example, the parvovirus template can be provided by aparvovirus particle or a second recombinant adenovirus particle.

According to the foregoing methods, the hybrid adenovirus vectortypically comprises the adenovirus 5′ and 3′ cis sequences sufficientfor adenovirus replication and packaging (i.e., the adenovirus terminalrepeats and PAC sequence). The parvovirus rep/cap sequences and, ifpresent, the AAV template are embedded in the adenovirus backbone andare flanked by the 5′ and 3′ cis sequences, so that these sequences maybe packaged into adenovirus capsids. As described above, the adenovirushelper sequences and the parvovirus rep/cap sequences are generally notflanked by ITRs so that these sequences are not packaged into theparvovirus virions.

Zhang et al., ((2001) Gene Ther. 18:704-12) describe a chimeric helpercomprising both adenovirus and the AAV rep and cap genes.

Herpesvirus may also be used as a helper virus in parvovirus packagingmethods. Hybrid herpesviruses encoding the parvovirus Rep protein(s) mayadvantageously facilitate scalable parvovirus vector production schemes.A hybrid herpes simplex virus type I (HSV-1) vector expressing the AAV-2rep and cap genes has been described (Conway et al., (1999) Gene Ther.6:986 and WO 00/17377.

As a further alternative, the virus vectors of the invention can beproduced in insect cells using baculovirus vectors to deliver therep/cap genes and parvovirus template as described, for example, byUrabe et al., (2002) Human Gene Ther. 13:1935-43.

Parvovirus vector stocks free of contaminating helper virus may beobtained by any method known in the art. For example, parvovirus andhelper virus may be readily differentiated based on size. Parvovirus mayalso be separated away from helper virus based on affinity for a heparinsubstrate (Zolotukhin et al., (1999) Gene Therapy 6:973). Deletedreplication-defective helper viruses can be used so that anycontaminating helper virus is not replication competent. As a furtheralternative, an adenovirus helper lacking late gene expression may beemployed, as only adenovirus early gene expression is required tomediate packaging of parvovirus. Adenovirus mutants defective for lategene expression are known in the art (e.g., ts100K and ts149 adenovirusmutants).

Recombinant Virus Vectors

The virus vectors of the present invention are useful for the deliveryof nucleic acids to cells in vitro, ex vivo, and in vivo. In particular,the virus vectors can be advantageously employed to deliver or transfernucleic acids to animal, including mammalian, cells.

Any heterologous nucleic acid sequence(s) of interest may be deliveredin the virus vectors of the present invention. Nucleic acids of interestinclude nucleic acids encoding polypeptides, including therapeutic(e.g., for medical or veterinary uses), immunogenic (e.g., forvaccines), or diagnostic polypeptides.

Therapeutic polypeptides include, but are not limited to, cysticfibrosis transmembrane regulator protein (CFTR), dystrophin (includingmini- and micro-dystrophins (see, e.g., Vincent et al., (1993) NatureGenetics 5:130; U.S. Patent Publication No. 2003/017131; Internationalpublication WO/2008/088895, Wang et al., Proc. Natl. Acad. Sci. USA97:13714-13719 (2000); and Gregorevic et al., Mol. Ther. 16:657-64(2008)), myostatin propeptide, follistatin, activin type II solublereceptor, IGF-1, anti-inflammatory polypeptides such as the Ikappa Bdominant mutant, sarcospan, utrophin (Tinsley et al., (1996) Nature384:349), mini-utrophin, clotting factors (e.g., Factor VIII, Factor IX,Factor X, etc.), erythropoietin, angiostatin, endostatin, catalase,tyrosine hydroxylase, superoxide dismutase, leptin, the LDL receptor,lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin,spectrin, α₁-antitrypsin, adenosine deaminase, hypoxanthine guaninephosphoribosyl transferase, β-glucocerebrosidase, sphingomyelinase,lysosomal hexosaminidase A, branched-chain keto acid dehydrogenase, RP65protein, cytokines (e.g., α-interferon, β-interferon, interferon-γ,interleukin-2, interleukin-4, granulocyte-macrophage colony stimulatingfactor, lymphotoxin, and the like), peptide growth factors, neurotrophicfactors and hormones (e.g., somatotropin, insulin, insulin-like growthfactors 1 and 2, platelet derived growth factor, epidermal growthfactor, fibroblast growth factor, nerve growth factor, neurotrophicfactor-3 and -4, brain-derived neurotrophic factor, bone morphogenicproteins [including RANKL and VEGF], glial derived growth factor,transforming growth factor-α and -β, and the like), lysosomal acidα-glucosidase, α-galactosidase A, receptors (e.g., the tumor necrosisgrowth factorα soluble receptor), S100A1, parvalbumin, adenylyl cyclasetype 6, a molecule that effects G-protein coupled receptor kinase type 2knockdown such as a truncated constitutively active bARKct,anti-inflammatory factors such as IRAP, anti-myostatin proteins,aspartoacylase, and monoclonal antibodies (including single chainmonoclonal antibodies; an exemplary Mab is the Herceptin® Mab). Otherillustrative heterologous nucleic acid sequences encode suicide geneproducts (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin,and tumor necrosis factor), proteins conferring resistance to a drugused in cancer therapy, tumor suppressor gene products (e.g., p53, Rb,Wt-1), TRAIL, FAS-ligand, and any other polypeptide that has atherapeutic effect in a subject in need thereof. Parvovirus vectors canalso be used to deliver monoclonal antibodies and antibody fragments,for example, an antibody or antibody fragment directed against myostatin(see, e.g., Fang et al., Nature Biotechnol. 23:584-590 (2005)).

Heterologous nucleic acid sequences encoding polypeptides include thoseencoding reporter polypeptides (e.g., an enzyme). Reporter polypeptidesare known in the art and include, but are not limited to, GreenFluorescent Protein, β-galactosidase, alkaline phosphatase, luciferase,and chloramphenicol acetyltransferase gene.

Alternatively, in particular embodiments of this invention, theheterologous nucleic acid may encode an antisense nucleic acid, aribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs thateffect spliceosome-mediated trans-splicing (see, Puttaraju et al.,(1999) Nature Biotech. 17:246; U.S. Pat. Nos. 6,013,487; 6,083,702),interfering RNAs (RNAi) including siRNA, shRNA or miRNA that mediategene silencing (see, Sharp et al., (2000) Science 287:2431), and othernon-translated RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc.Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.),and the like. Exemplary untranslated RNAs include RNAi against amultiple drug resistance (MDR) gene product (e.g., to treat and/orprevent tumors and/or for administration to the heart to prevent damageby chemotherapy), RNAi against myostatin (e.g., for Duchenne musculardystrophy), RNAi against VEGF (e.g., to treat and/or prevent tumors),RNAi against phospholamban (e.g., to treat cardiovascular disease, see,e.g., Andino et al., J. Gene Med. 10:132-142 (2008) and Li et al., ActaPharmacol Sin. 26:51-55 (2005)); phospholamban inhibitory ordominant-negative molecules such as phospholamban S16E (e.g., to treatcardiovascular disease, see, e.g., Hoshijima et al., Nat. Med. 8:864-871(2002)), RNAi to adenosine kinase (e.g., for epilepsy), RNAi to asarcoglycan [e.g., α, β, γ], RNAi against myostatin, myostatinpropeptide, follistatin, or activin type II soluble receptor, RNAiagainst anti-inflammatory polypeptides such as the Ikappa B dominantmutant, and RNAi directed against pathogenic organisms and viruses(e.g., hepatitis B virus, human immunodeficiency virus, CMV, herpessimplex virus, human papilloma virus, etc.).

Alternatively, in particular embodiments of this invention, theheterologous nucleic acid may encode protein phosphatase inhibitor I(I-1), serca2a, zinc finger proteins that regulate the phospholambangene, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase(BARK), phosphoinositide-3 kinase (PI3 kinase), a molecule that effectsG-protein coupled receptor kinase type 2 knockdown such as a truncatedconstitutively active bARKct; calsarcin, RNAi against phospholamban;phospholamban inhibitory or dominant-negative molecules such asphospholamban S16E, enos, inos, or bone morphogenic proteins (includingBNP 2, 7, etc., RANKL and/or VEGF).

The virus vector may also comprise a heterologous nucleic acid thatshares homology with and recombines with a locus on a host chromosome.This approach can be utilized, for example, to correct a genetic defectin the host cell.

The present invention also provides virus vectors that express animmunogenic polypeptide, e.g., for vaccination. The nucleic acid mayencode any immunogen of interest known in the art including, but notlimited to, immunogens from human immunodeficiency virus (HIV), simianimmunodeficiency virus (SIV), influenza virus, HIV or SIV gag proteins,tumor antigens, cancer antigens, bacterial antigens, viral antigens, andthe like.

The use of parvoviruses as vaccine vectors is known in the art (see,e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S.Pat. No. 5,916,563 to Young et al., U.S. Pat. No. 5,905,040 to Mazzaraet al., U.S. Pat. Nos. 5,882,652, 5,863,541 to Samulski et al.). Theantigen may be presented in the parvovirus capsid. Alternatively, theantigen may be expressed from a heterologous nucleic acid introducedinto a recombinant vector genome. Any immunogen of interest as describedherein and/or as is known in the art can be provided by the virus vectorof the present invention.

An immunogenic polypeptide can be any polypeptide suitable for elicitingan immune response and/or protecting the subject against an infectionand/or disease, including, but not limited to, microbial, bacterial,protozoal, parasitic, fungal and/or viral infections and diseases. Forexample, the immunogenic polypeptide can be an orthomyxovirus immunogen(e.g., an influenza virus immunogen, such as the influenza virushemagglutinin (HA) surface protein or the influenza virus nucleoprotein,or an equine influenza virus immunogen) or a lentivirus immunogen (e.g.,an equine infectious anemia virus immunogen, a Simian ImmunodeficiencyVirus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV)immunogen, such as the HIV or SIV envelope GP160 protein, the HIV or SIVmatrix/capsid proteins, and the HIV or SIV gag, pol and env genesproducts). The immunogenic polypeptide can also be an arenavirusimmunogen (e.g., Lassa fever virus immunogen, such as the Lassa fevervirus nucleocapsid protein and the Lassa fever envelope glycoprotein), apoxvirus immunogen (e.g., a vaccinia virus immunogen, such as thevaccinia L1 or L8 gene products), a flavivirus immunogen (e.g., a yellowfever virus immunogen or a Japanese encephalitis virus immunogen), afilovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virusimmunogen, such as NP and GP gene products), a bunyavirus immunogen(e.g., RVFV, CCHF, and/or SFS virus immunogens), or a coronavirusimmunogen (e.g., an infectious human coronavirus immunogen, such as thehuman coronavirus envelope glycoprotein, or a porcine transmissiblegastroenteritis virus immunogen, or an avian infectious bronchitis virusimmunogen). The immunogenic polypeptide can further be a polioimmunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens) a mumpsimmunogen, a measles immunogen, a rubella immunogen, a diphtheria toxinor other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g.,hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and/or any othervaccine immunogen now known in the art or later identified as animmunogen.

Alternatively, the immunogenic polypeptide can be any tumor or cancercell antigen. Optionally, the tumor or cancer antigen is expressed onthe surface of the cancer cell. Exemplary cancer and tumor cell antigensare described in S. A. Rosenberg (Immunity 10:281 (1991)). Otherillustrative cancer and tumor antigens include, but are not limited to:BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2,BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, Caspase-8,KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigens (Kawakami etal., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994)J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124),MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15,tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2/neugene product (U.S. Pat. No. 4,968,603), CA 125, LK26, FB5 (endosialin),TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN(sialyl Tn antigen), c-erbB-2 proteins, PSA, L-CanAg, estrogen receptor,milk fat globulin, p53 tumor suppressor protein (Levine, (1993) Ann.Rev. Biochem. 62:623); mucin antigens (International Patent PublicationNo. WO 90/05142); telomerases; nuclear matrix proteins; prostatic acidphosphatase; papilloma virus antigens; and/or antigens now known orlater discovered to be associated with the following cancers: melanoma,adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma,Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer,leukemia, uterine cancer, breast cancer, prostate cancer, ovariancancer, cervical cancer, bladder cancer, kidney cancer, pancreaticcancer, brain cancer and any other cancer or malignant condition nowknown or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med.47:481-91).

As a further alternative, the heterologous nucleic acid can encode anypolypeptide that is desirably produced in a cell in vitro, ex vivo, orin vivo. For example, the virus vectors may be introduced into culturedcells and the expressed gene product isolated therefrom.

It will be understood by those skilled in the art that the heterologousnucleic acid(s) of interest can be operably associated with appropriatecontrol sequences. For example, the heterologous nucleic acid can beoperably associated with expression control elements, such astranscription/translation control signals, origins of replication,polyadenylation signals, internal ribosome entry sites (IRES),promoters, and/or enhancers, and the like.

Those skilled in the art will appreciate that a variety ofpromoter/enhancer elements can be used depending on the level andtissue-specific expression desired. The promoter/enhancer can beconstitutive or inducible, depending on the pattern of expressiondesired. The promoter/enhancer can be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced.

In particular embodiments, the promoter/enhancer elements can be nativeto the target cell or subject to be treated. In representativeembodiments, the promoters/enhancer element can be native to theheterologous nucleic acid sequence. The promoter/enhancer element isgenerally chosen so that it functions in the target cell(s) of interest.Further, in particular embodiments the promoter/enhancer element is amammalian promoter/enhancer element. The promoter/enhancer element maybe constitutive or inducible.

Inducible expression control elements are typically advantageous inthose applications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery can be tissue-specific or-preferred promoter/enhancer elements, and include muscle specific orpreferred (including cardiac, skeletal and/or smooth muscle specific orpreferred), neural tissue specific or preferred (includingbrain-specific or preferred), eye specific or preferred (includingretina-specific and cornea-specific), liver specific or preferred, bonemarrow specific or preferred, pancreatic specific or preferred, spleenspecific or preferred, and lung specific or preferred promoter/enhancerelements. Other inducible promoter/enhancer elements includehormone-inducible and metal-inducible elements. Exemplary induciblepromoters/enhancer elements include, but are not limited to, a Teton/off element, a RU486-inducible promoter, an ecdysone-induciblepromoter, a rapamycin-inducible promoter, and a metallothioneinpromoter.

In embodiments wherein the heterologous nucleic acid sequence(s) istranscribed and then translated in the target cells, specific initiationsignals are generally included for efficient translation of insertedprotein coding sequences. These exogenous translational controlsequences, which may include the ATG initiation codon and adjacentsequences, can be of a variety of origins, both natural and synthetic.

The virus vectors according to the present invention provide a means fordelivering heterologous nucleic acids into a broad range of cells,including dividing and non-dividing cells. The virus vectors can beemployed to deliver a nucleic acid of interest to a cell in vitro, e.g.,to produce a polypeptide in vitro or for ex vivo gene therapy. The virusvectors are additionally useful in a method of delivering a nucleic acidto a subject in need thereof, e.g., to express an immunogenic ortherapeutic polypeptide or a functional RNA. In this manner, thepolypeptide or functional RNA can be produced in vivo in the subject.The subject can be in need of the polypeptide because the subject has adeficiency of the polypeptide. Further, the method can be practicedbecause the production of the polypeptide or functional RNA in thesubject may impart some beneficial effect.

The virus vectors can also be used to produce a polypeptide of interestor functional RNA in cultured cells or in a subject (e.g., using thesubject as a bioreactor to produce the polypeptide or to observe theeffects of the functional RNA on the subject, for example, in connectionwith screening methods).

In general, the virus vectors of the present invention can be employedto deliver a heterologous nucleic acid encoding a polypeptide orfunctional RNA to treat and/or prevent any disease state for which it isbeneficial to deliver a therapeutic polypeptide or functional RNA.Illustrative disease states include, but are not limited to: cysticfibrosis (cystic fibrosis transmembrane regulator protein) and otherdiseases of the lung, hemophilia A (Factor VIII), hemophilia B (FactorIX), thalassemia (β-globin), anemia (erythropoietin) and other blooddisorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis(β-interferon), Parkinson's disease (glial-cell line derivedneurotrophic factor [GDNF]), Huntington's disease (RNAi to removerepeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophicfactors), and other neurological disorders, cancer (endostatin,angiostatin, TRAIL, FAS-ligand, cytokines including interferons; RNAiincluding RNAi against VEGF or the multiple drug resistance geneproduct), diabetes mellitus (insulin), muscular dystrophies includingDuchenne (dystrophin, mini-dystrophin, insulin-like growth factor I, asarcoglycan [e.g., α, β, γ], RNAi against myostatin, myostatinpropeptide, follistatin, activin type II soluble receptor,anti-inflammatory polypeptides such as the Ikappa B dominant mutant,sarcospan, utrophin, mini-utrophin, RNAi against splice junctions in thedystrophin gene to induce exon skipping [see, e.g., WO/2003/095647],antisense against U7 snRNAs to induce exon skipping [see, e.g.,WO/2006/021724], and antibodies or antibody fragments against myostatinor myostatin propeptide) and Becker, Gaucher disease(glucocerebrosidase), Hurler's disease (α-L-iduronidase), adenosinedeaminase deficiency (adenosine deaminase), glycogen storage diseases(e.g., Fabry disease [α-galactosidase] and Pompe disease [lysosomal acidα-glucosidase]) and other metabolic defects, congenital emphysema(α1-antitrypsin), Lesch-Nyhan Syndrome (hypoxanthine guaninephosphoribosyl transferase), Niemann-Pick disease (sphingomyelinase),Tays Sachs disease (lysosomal hexosaminidase A), Maple Syrup UrineDisease (branched-chain keto acid dehydrogenase), retinal degenerativediseases (and other diseases of the eye and retina; e.g., PDGF formacular degeneration), diseases of solid organs such as brain (includingParkinson's Disease [GDNF], astrocytomas [endostatin, angiostatin and/orRNAi against VEGF], glioblastomas [endostatin, angiostatin and/or RNAiagainst VEGF]), liver, kidney, heart including congestive heart failureor peripheral artery disease (PAD) (e.g., by delivering proteinphosphatase inhibitor I (I-1), serca2a, zinc finger proteins thatregulate the phospholamban gene, Barkct, β2-adrenergic receptor,β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3kinase), S100A1, parvalbumin, adenylyl cyclase type 6, a molecule thateffects G-protein coupled receptor kinase type 2 knockdown such as atruncated constitutively active bARKct; calsarcin, RNAi againstphospholamban; phospholamban inhibitory or dominant-negative moleculessuch as phospholamban S16E, etc.), arthritis (insulin-like growthfactors), joint disorders (insulin-like growth factor 1 and/or 2),intimal hyperplasia (e.g., by delivering enos, inos), improve survivalof heart transplants (superoxide dismutase), AIDS (soluble CD4), musclewasting (insulin-like growth factor I), kidney deficiency(erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatoryfactors such as IRAP and TNFα soluble receptor), hepatitis(α-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia(ornithine transcarbamylase), Krabbe's disease (galactocerebrosidase),Batten's disease, spinal cerebral ataxias including SCA1, SCA2 and SCA3,phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, andthe like. The invention can further be used following organtransplantation to increase the success of the transplant and/or toreduce the negative side effects of organ transplantation or adjuncttherapies (e.g., by administering immunosuppressant agents or inhibitorynucleic acids to block cytokine production). As another example, bonemorphogenic proteins (including BNP 2, 7, etc., RANKL and/or VEGF) canbe administered with a bone allograft, for example, following a break orsurgical removal in a cancer patient.

Gene transfer has substantial potential use for understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Ingeneral, the above disease states fall into two classes: deficiencystates, usually of enzymes, which are generally inherited in a recessivemanner, and unbalanced states, which may involve regulatory orstructural proteins, and which are typically inherited in a dominantmanner. For deficiency state diseases, gene transfer can be used tobring a normal gene into affected tissues for replacement therapy, aswell as to create animal models for the disease using antisensemutations. For unbalanced disease states, gene transfer can be used tocreate a disease state in a model system, which can then be used inefforts to counteract the disease state. Thus, virus vectors accordingto the present invention permit the treatment and/or prevention ofgenetic diseases.

The virus vectors according to the present invention may also beemployed to provide a functional RNA to a cell in vitro or in vivo.Expression of the functional RNA in the cell, for example, can diminishexpression of a particular target protein by the cell. Accordingly,functional RNA can be administered to decrease expression of aparticular protein in a subject in need thereof. Functional RNA can alsobe administered to cells in vitro to regulate gene expression and/orcell physiology, e.g., to optimize cell or tissue culture systems or inscreening methods.

Virus vectors according to the instant invention find use in diagnosticand screening methods, whereby a nucleic acid of interest is transientlyor stably expressed in a cell culture system, or alternatively, atransgenic animal model.

The virus vectors of the present invention can also be used for variousnon-therapeutic purposes, including but not limited to use in protocolsto assess gene targeting, clearance, transcription, translation, etc.,as would be apparent to one skilled in the art. The virus vectors canalso be used for the purpose of evaluating safety (spread, toxicity,immunogenicity, etc.). Such data, for example, are considered by theUnited States Food and Drug Administration as part of the regulatoryapproval process prior to evaluation of clinical efficacy.

As a further aspect, the virus vectors of the present invention may beused to produce an immune response in a subject. According to thisembodiment, a virus vector comprising a heterologous nucleic acidsequence encoding an immunogenic polypeptide can be administered to asubject, and an active immune response is mounted by the subject againstthe immunogenic polypeptide. Immunogenic polypeptides are as describedhereinabove. In some embodiments, a protective immune response iselicited.

Alternatively, the virus vector may be administered to a cell ex vivoand the altered cell is administered to the subject. The virus vectorcomprising the heterologous nucleic acid is introduced into the cell,and the cell is administered to the subject, where the heterologousnucleic acid encoding the immunogen can be expressed and induce animmune response in the subject against the immunogen. In particularembodiments, the cell is an antigen-presenting cell (e.g., a dendriticcell).

An “active immune response” or “active immunity” is characterized by“participation of host tissues and cells after an encounter with theimmunogen. It involves differentiation and proliferation ofimmunocompetent cells in lymphoreticular tissues, which lead tosynthesis of antibody or the development of cell-mediated reactivity, orboth.” Herbert B. Herscowitz, Immunophysiology: Cell Function andCellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, anactive immune response is mounted by the host after exposure to animmunogen by infection or by vaccination. Active immunity can becontrasted with passive immunity, which is acquired through the“transfer of preformed substances (antibody, transfer factor, thymicgraft, interleukin-2) from an actively immunized host to a non-immunehost.” Id.

A “protective” immune response or “protective” immunity as used hereinindicates that the immune response confers some benefit to the subjectin that it prevents or reduces the incidence of disease. Alternatively,a protective immune response or protective immunity may be useful in thetreatment and/or prevention of disease, in particular cancer or tumors(e.g., by preventing cancer or tumor formation, by causing regression ofa cancer or tumor and/or by preventing metastasis and/or by preventinggrowth of metastatic nodules). The protective effects may be complete orpartial, as long as the benefits of the treatment outweigh anydisadvantages thereof.

In particular embodiments, the virus vector or cell comprising theheterologous nucleic acid can be administered in an immunogenicallyeffective amount, as described below.

The virus vectors of the present invention can also be administered forcancer immunotherapy by administration of a virus vector expressing oneor more cancer cell antigens (or an immunologically similar molecule) orany other immunogen that produces an immune response against a cancercell. To illustrate, an immune response can be produced against a cancercell antigen in a subject by administering a virus vector comprising aheterologous nucleic acid encoding the cancer cell antigen, for exampleto treat a patient with cancer and/or to prevent cancer from developingin the subject. The virus vector may be administered to a subject invivo or by using ex vivo methods, as described herein. Alternatively,the cancer antigen can be expressed as part of the virus capsid or beotherwise associated with the virus capsid as described above.

As another alternative, any other therapeutic nucleic acid (e.g., RNAi)or polypeptide (e.g., cytokine) known in the art can be administered totreat and/or prevent cancer.

As used herein, the term “cancer” encompasses tumor-forming cancers.Likewise, the term “cancerous tissue” encompasses tumors. A “cancer cellantigen” encompasses tumor antigens.

The term “cancer” has its understood meaning in the art, for example, anuncontrolled growth of tissue that has the potential to spread todistant sites of the body (i.e., metastasize). Exemplary cancersinclude, but are not limited to melanoma, adenocarcinoma, thymoma,lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma,lung cancer, liver cancer, colon cancer, leukemia, uterine cancer,breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladdercancer, kidney cancer, pancreatic cancer, brain cancer and any othercancer or malignant condition now known or later identified. Inrepresentative embodiments, the invention provides a method of treatingand/or preventing tumor-forming cancers.

The term “tumor” is also understood in the art, for example, as anabnormal mass of undifferentiated cells within a multicellular organism.Tumors can be malignant or benign. In representative embodiments, themethods disclosed herein are used to prevent and treat malignant tumors.

By the terms “treating cancer,” “treatment of cancer” and equivalentterms it is intended that the severity of the cancer is reduced or atleast partially eliminated and/or the progression of the disease isslowed and/or controlled and/or the disease is stabilized. In particularembodiments, these terms indicate that metastasis of the cancer isprevented or reduced or at least partially eliminated and/or that growthof metastatic nodules is prevented or reduced or at least partiallyeliminated.

By the terms “prevention of cancer” or “preventing cancer” andequivalent terms it is intended that the methods at least partiallyeliminate or reduce and/or delay the incidence and/or severity of theonset of cancer. Alternatively stated, the onset of cancer in thesubject may be reduced in likelihood or probability and/or delayed.

In particular embodiments, cells may be removed from a subject withcancer and contacted with a virus vector according to the instantinvention. The modified cell is then administered to the subject,whereby an immune response against the cancer cell antigen is elicited.This method can be advantageously employed with immunocompromisedsubjects that cannot mount a sufficient immune response in vivo (i.e.,cannot produce enhancing antibodies in sufficient quantities).

It is known in the art that immune responses may be enhanced byimmunomodulatory cytokines (e.g., α-interferon, β-interferon,γ-interferon, ω-interferon, τ-interferon, interleukin-1α,interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin5, interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin 12, interleukin-13,interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumornecrosis factor-α, tumor necrosis factor-β, monocyte chemoattractantprotein-1, granulocyte-macrophage colony stimulating factor, andlymphotoxin). Accordingly, immunomodulatory cytokines (preferably, CTLinductive cytokines) may be administered to a subject in conjunctionwith the virus vector.

Cytokines may be administered by any method known in the art. Exogenouscytokines may be administered to the subject, or alternatively, anucleic acid encoding a cytokine may be delivered to the subject using asuitable vector, and the cytokine produced in vivo.

Subjects, Pharmaceutical Formulations, and Modes of Administration

Virus vectors and capsids according to the present invention find use inboth veterinary and medical applications. Suitable subjects include bothavians and mammals. The term “avian” as used herein includes, but is notlimited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots,parakeets, and the like. The term “mammal” as used herein includes, butis not limited to, humans, non-human primates, bovines, ovines,caprines, equines, felines, canines, lagomorphs, etc. Human subjectsinclude neonates, infants, juveniles and adults.

In particular embodiments, the present invention provides apharmaceutical composition comprising a virus vector and/or capsid ofthe invention in a pharmaceutically acceptable carrier and, optionally,other medicinal agents, pharmaceutical agents, stabilizing agents,buffers, carriers, adjuvants, diluents, etc. For injection, the carrierwill typically be a liquid. For other methods of administration, thecarrier may be either solid or liquid. For inhalation administration,the carrier will be respirable, and optionally can be in solid or liquidparticulate form.

By “pharmaceutically acceptable” it is meant a material that is nottoxic or otherwise undesirable, i.e., the material may be administeredto a subject without causing any undesirable biological effects.

One aspect of the present invention is a method of transferring anucleic acid to a cell in vitro. The virus vector may be introduced intothe cells at the appropriate multiplicity of infection according tostandard transduction methods suitable for the particular target cells.Titers of virus vector to administer can vary, depending upon the targetcell type and number, and the particular virus vector, and can bedetermined by those of skill in the art without undue experimentation.In representative embodiments, at least about 10³ infectious units, morepreferably at least about 10⁵ infectious units are introduced to thecell.

The cell(s) into which the virus vector is introduced can be of anytype, including but not limited to neural cells (including cells of theperipheral and central nervous systems, in particular, brain cells suchas neurons and oligodendrocytes), lung cells, cells of the eye(including retinal cells, retinal pigment epithelium, and cornealcells), blood vessel cells (e.g., endothelial cells, intimal cells),epithelial cells (e.g., gut and respiratory epithelial cells), musclecells (e.g., skeletal muscle cells, cardiac muscle cells, smooth musclecells and/or diaphragm muscle cells), dendritic cells, pancreatic cells(including islet cells), hepatic cells, kidney cells, myocardial cells,bone cells (e.g., bone marrow stem cells), hematopoietic stem cells,spleen cells, keratinocytes, fibroblasts, endothelial cells, prostatecells, germ cells, and the like. In representative embodiments, the cellcan be any progenitor cell. As a further possibility, the cell can be astem cell (e.g., neural stem cell, liver stem cell). As still a furtheralternative, the cell can be a cancer or tumor cell. Moreover, the cellcan be from any species of origin, as indicated above.

The virus vector can be introduced into cells in vitro for the purposeof administering the modified cell to a subject. In particularembodiments, the cells have been removed from a subject, the virusvector is introduced therein, and the cells are then administered backinto the subject. Methods of removing cells from subject formanipulation ex vivo, followed by introduction back into the subject areknown in the art (see, e.g., U.S. Pat. No. 5,399,346). Alternatively,the recombinant virus vector can be introduced into cells from a donorsubject, into cultured cells, or into cells from any other suitablesource, and the cells are administered to a subject in need thereof(i.e., a “recipient” subject).

Suitable cells for ex vivo gene delivery are as described above. Dosagesof the cells to administer to a subject will vary upon the age,condition and species of the subject, the type of cell, the nucleic acidbeing expressed by the cell, the mode of administration, and the like.Typically, at least about 10² to about 10⁸ cells or at least about 10³to about 10⁶ cells will be administered per dose in a pharmaceuticallyacceptable carrier. In particular embodiments, the cells transduced withthe virus vector are administered to the subject in a treatmenteffective or prevention effective amount in combination with apharmaceutical carrier.

In some embodiments, the virus vector is introduced into a cell and thecell can be administered to a subject to elicit an immunogenic responseagainst the delivered polypeptide (e.g., expressed as a transgene or inthe capsid). Typically, a quantity of cells expressing animmunogenically effective amount of the polypeptide in combination witha pharmaceutically acceptable carrier is administered. An“immunogenically effective amount” is an amount of the expressedpolypeptide that is sufficient to evoke an active immune responseagainst the polypeptide in the subject to which the pharmaceuticalformulation is administered. In particular embodiments, the dosage issufficient to produce a protective immune response (as defined above).The degree of protection conferred need not be complete or permanent, aslong as the benefits of administering the immunogenic polypeptideoutweigh any disadvantages thereof.

A further aspect of the invention is a method of administering the virusvector to subjects. Administration of the virus vectors and/or capsidsaccording to the present invention to a human subject or an animal inneed thereof can be by any means known in the art. Optionally, the virusvector and/or capsid is delivered in a treatment effective or preventioneffective dose in a pharmaceutically acceptable carrier.

The virus vectors and/or capsids of the invention can further beadministered to elicit an immunogenic response (e.g., as a vaccine).Typically, immunogenic compositions of the present invention comprise animmunogenically effective amount of virus vector and/or capsid incombination with a pharmaceutically acceptable carrier. Optionally, thedosage is sufficient to produce a protective immune response (as definedabove). The degree of protection conferred need not be complete orpermanent, as long as the benefits of administering the immunogenicpolypeptide outweigh any disadvantages thereof. Subjects and immunogensare as described above.

Dosages of the virus vector and/or capsid to be administered to asubject depend upon the mode of administration, the disease or conditionto be treated and/or prevented, the individual subject's condition, theparticular virus vector or capsid, and the nucleic acid to be delivered,and the like, and can be determined in a routine manner. Exemplary dosesfor achieving therapeutic effects are titers of at least about 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵ transducing units,optionally about 10⁸-10¹³ transducing units.

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) may be employed to achieve thedesired level of gene expression over a period of various intervals,e.g., daily, weekly, monthly, yearly, etc.

Exemplary modes of administration include oral, rectal, transmucosal,intranasal, inhalation (e.g., via an aerosol), buccal (e.g.,sublingual), vaginal, intrathecal, intraocular, transdermal,intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous,subcutaneous, intradermal, intracranial, intramuscular [includingadministration to skeletal, diaphragm and/or cardiac muscle],intrapleural, intracerebral, and intraarticular), topical (e.g., to bothskin and mucosal surfaces, including airway surfaces, and transdermaladministration), intralymphatic, and the like, as well as direct tissueor organ injection (e.g., to liver, eye [including intravitreal andsubretinal], skeletal muscle, cardiac muscle, diaphragm muscle orbrain).

Administration can be to any site in a subject, including, withoutlimitation, a site selected from the group consisting of the brain, askeletal muscle, a smooth muscle, the heart, the diaphragm, the airwayepithelium, the liver, the kidney, the spleen, the pancreas, the skin,and the eye.

Administration can also be to a tumor (e.g., in or near a tumor or alymph node). The most suitable route in any given case will depend onthe nature and severity of the condition being treated and/or preventedand on the nature of the particular vector that is being used.

Administration to skeletal muscle according to the present inventionincludes but is not limited to administration to skeletal muscle in thelimbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back,neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/ordigits. Suitable skeletal muscles include but are not limited toabductor digiti minimi (in the hand), abductor digiti minimi (in thefoot), abductor hallucis, abductor ossis metatarsi quinti, abductorpollicis brevis, abductor pollicis longus, adductor brevis, adductorhallucis, adductor longus, adductor magnus, adductor pollicis, anconeus,anterior scalene, articularis genus, biceps brachii, biceps femoris,brachialis, brachioradialis, buccinator, coracobrachialis, corrugatorsupercilii, deltoid, depressor anguli oris, depressor labii inferioris,digastric, dorsal interossei (in the hand), dorsal interossei (in thefoot), extensor carpi radialis brevis, extensor carpi radialis longus,extensor carpi ulnaris, extensor digiti minimi, extensor digitorum,extensor digitorum brevis, extensor digitorum longus, extensor hallucisbrevis, extensor hallucis longus, extensor indicis, extensor pollicisbrevis, extensor pollicis longus, flexor carpi radialis, flexor carpiulnaris, flexor digiti minimi brevis (in the hand), flexor digiti minimibrevis (in the foot), flexor digitorum brevis, flexor digitorum longus,flexor digitorum profundus, flexor digitorum superficialis, flexorhallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexorpollicis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus,gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis,iliocostalis lumborum, iliocostalis thoracis, illiacus, inferiorgemellus, inferior oblique, inferior rectus, infraspinatus,interspinalis, intertransversi, lateral pterygoid, lateral rectus,latissimus dorsi, levator anguli oris, levator labii superioris, levatorlabii superioris alaeque nasi, levator palpebrae superioris, levatorscapulae, long rotators, longissimus capitis, longissimus cervicis,longissimus thoracis, longus capitis, longus colli, lumbricals (in thehand), lumbricals (in the foot), masseter, medial pterygoid, medialrectus, middle scalene, multifidus, mylohyoid, obliquus capitisinferior, obliquus capitis superior, obturator externus, obturatorinternus, occipitalis, omohyoid, opponens digiti minimi, opponenspollicis, orbicularis oculi, orbicularis oris, palmar interossei,palmaris brevis, palmaris longus, pectineus, pectoralis major,pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius,piriformis, plantar interossei, plantaris, platysma, popliteus,posterior scalene, pronator quadratus, pronator teres, psoas major,quadratus femoris, quadratus plantae, rectus capitis anterior, rectuscapitis lateralis, rectus capitis posterior major, rectus capitisposterior minor, rectus femoris, rhomboid major, rhomboid minor,risorius, sartorius, scalenus minimus, semimembranosus, semispinaliscapitis, semispinalis cervicis, semispinalis thoracis, semitendinosus,serratus anterior, short rotators, soleus, spinalis capitis, spinaliscervicis, spinalis thoracis, splenius capitis, splenius cervicis,sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius,subscapularis, superior gemellus, superior oblique, superior rectus,supinator, supraspinatus, temporalis, tensor fascia lata, teres major,teres minor, thoracis, thyrohyoid, tibialis anterior, tibialisposterior, trapezius, triceps brachii, vastus intermedius, vastuslateralis, vastus medialis, zygomaticus major, and zygomaticus minor,and any other suitable skeletal muscle as known in the art.

The virus vector can be delivered to skeletal muscle by intravenousadministration, intra-arterial administration, intraperitonealadministration, limb perfusion, (optionally, isolated limb perfusion ofa leg and/or arm; see, e.g. Arruda et al., (2005) Blood 105: 3458-3464),and/or direct intramuscular injection. In particular embodiments, thevirus vector and/or capsid is administered to a limb (arm and/or leg) ofa subject (e.g., a subject with muscular dystrophy such as DMD) by limbperfusion, optionally isolated limb perfusion (e.g., by intravenous orintra-articular administration. In embodiments of the invention, thevirus vectors and/or capsids of the invention can advantageously beadministered without employing “hydrodynamic” techniques. Tissuedelivery (e.g., to muscle) of prior art vectors is often enhanced byhydrodynamic techniques (e.g., intravenous/intravenous administration ina large volume), which increase pressure in the vasculature andfacilitate the ability of the vector to cross the endothelial cellbarrier. In particular embodiments, the viral vectors and/or capsids ofthe invention can be administered in the absence of hydrodynamictechniques such as high volume infusions and/or elevated intravascularpressure (e.g., greater than normal systolic pressure, for example, lessthan or equal to a 5%, 10%, 15%, 20%, 25% increase in intravascularpressure over normal systolic pressure). Such methods may reduce oravoid the side effects associated with hydrodynamic techniques such asedema, nerve damage and/or compartment syndrome.

Administration to cardiac muscle includes administration to the leftatrium, right atrium, left ventricle, right ventricle and/or septum. Thevirus vector and/or capsid can be delivered to cardiac muscle byintravenous administration, intra-arterial administration such asintra-aortic administration, direct cardiac injection (e.g., into leftatrium, right atrium, left ventricle, right ventricle), and/or coronaryartery perfusion.

Administration to diaphragm muscle can be by any suitable methodincluding intravenous administration, intra-arterial administration,and/or intra-peritoneal administration.

Administration to smooth muscle can be by any suitable method includingintravenous administration, intra-arterial administration, and/orintra-peritoneal administration. In one embodiment, administration canbe to endothelial cells present in, near, and/or on smooth muscle.

Delivery to a target tissue can also be achieved by delivering a depotcomprising the virus vector and/or capsid. In representativeembodiments, a depot comprising the virus vector and/or capsid isimplanted into skeletal, smooth, cardiac and/or diaphragm muscle tissueor the tissue can be contacted with a film or other matrix comprisingthe virus vector and/or capsid. Such implantable matrices or substratesare described in U.S. Pat. No. 7,201,898.

In particular embodiments, a virus vector according to the presentinvention is administered to skeletal muscle, diaphragm muscle and/orcardiac muscle (e.g., to treat and/or prevent muscular dystrophy orheart disease [for example, PAD or congestive heart failure]).

In representative embodiments, the invention is used to treat and/orprevent disorders of skeletal, cardiac and/or diaphragm muscle.

In a representative embodiment, the invention provides a method oftreating and/or preventing muscular dystrophy in a subject in needthereof, the method comprising: administering a treatment or preventioneffective amount of a virus vector of the invention to a mammaliansubject, wherein the virus vector comprises a heterologous nucleic acidencoding dystrophin, a mini-dystrophin, a micro-dystrophin, myostatinpropeptide, follistatin, activin type II soluble receptor, IGF-1,anti-inflammatory polypeptides such as the Ikappa B dominant mutant,sarcospan, utrophin, a micro-dystrophin, laminin-α2, α-sarcoglycan,β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IGF-1, an antibody orantibody fragment against myostatin or myostatin propeptide, and/or RNAiagainst myostatin. In particular embodiments, the virus vector can beadministered to skeletal, diaphragm and/or cardiac muscle as describedelsewhere herein.

Alternatively, the invention can be practiced to deliver a nucleic acidto skeletal, cardiac or diaphragm muscle, which is used as a platformfor production of a polypeptide (e.g., an enzyme) or functional RNA(e.g., RNAi, microRNA, antisense RNA) that normally circulates in theblood or for systemic delivery to other tissues to treat and/or preventa disorder (e.g., a metabolic disorder, such as diabetes (e.g.,insulin), hemophilia (e.g., Factor IX or Factor VIII), amucopolysaccharide disorder (e.g., Sly syndrome, Hurler Syndrome, ScheieSyndrome, Hurler-Scheie Syndrome, Hunter's Syndrome, Sanfilippo SyndromeA, B, C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome, etc.) or alysosomal storage disorder (such as Gaucher's disease[glucocerebrosidase], Pompe disease [lysosomal acid α-glucosidase] orFabry disease [α-galactosidase A]) or a glycogen storage disorder (suchas Pompe disease [lysosomal acid α glucosidase]). Other suitableproteins for treating and/or preventing metabolic disorders aredescribed above. The use of muscle as a platform to express a nucleicacid of interest is described in U.S. Patent Publication No.2002/0192189.

Thus, as one aspect, the invention further encompasses a method oftreating and/or preventing a metabolic disorder in a subject in needthereof, the method comprising: administering a treatment or preventioneffective amount of a virus vector of the invention to a subject (e.g.,to skeletal muscle of a subject), wherein the virus vector comprises aheterologous nucleic acid encoding a polypeptide, wherein the metabolicdisorder is a result of a deficiency and/or defect in the polypeptide.Illustrative metabolic disorders and heterologous nucleic acids encodingpolypeptides are described herein. Optionally, the polypeptide issecreted (e.g., a polypeptide that is a secreted polypeptide in itsnative state or that has been engineered to be secreted, for example, byoperable association with a secretory signal sequence as is known in theart). Without being limited by any particular theory of the invention,according to this embodiment, administration to the skeletal muscle canresult in secretion of the polypeptide into the systemic circulation anddelivery to target tissue(s). Methods of delivering virus vectors toskeletal muscle are described in more detail herein.

The invention can also be practiced to produce antisense RNA, RNAi orother functional RNA (e.g., a ribozyme) for systemic delivery.

The invention also provides a method of treating and/or preventingcongenital heart failure or PAD in a subject in need thereof, the methodcomprising administering a treatment or prevention effective amount of avirus vector of the invention to a mammalian subject, wherein the virusvector comprises a heterologous nucleic acid encoding, for example, asarcoplasmic endoreticulum Ca²⁺-ATPase (SERCA2a), an angiogenic factor,phosphatase inhibitor I (I-1), RNAi against phospholamban; aphospholamban inhibitory or dominant-negative molecule such asphospholamban S16E, a zinc finger protein that regulates thephospholamban gene, β2-adrenergic receptor, β2-adrenergic receptorkinase (BARK), PI3 kinase, calsarcan, a β-adrenergic receptor kinaseinhibitor (βARKct), inhibitor 1 of protein phosphatase 1, S100A1,parvalbumin, adenylyl cyclase type 6, a molecule that effects G-proteincoupled receptor kinase type 2 knockdown such as a truncatedconstitutively active bARKct, Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD,kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir-206 and/or mir-208.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one may administer the virus vector and/or virus capsids of theinvention in a local rather than systemic manner, for example, in adepot or sustained-release formulation. Further, the virus vector and/orvirus capsid can be delivered adhered to a surgically implantable matrix(e.g., as described in U.S. Patent Publication No. 2004-0013645).

The virus vectors disclosed herein can be administered to the lungs of asubject by any suitable means, optionally by administering an aerosolsuspension of respirable particles comprised of the virus vectors and/orvirus capsids, which the subject inhales. The respirable particles canbe liquid or solid. Aerosols of liquid particles comprising the virusvectors and/or virus capsids may be produced by any suitable means, suchas with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer,as is known to those of skill in the art. See, e.g., U.S. Pat. No.4,501,729. Aerosols of solid particles comprising the virus vectorsand/or capsids may likewise be produced with any solid particulatemedicament aerosol generator, by techniques known in the pharmaceuticalart.

The virus vectors can be administered to tissues of the CNS (e.g.,brain, eye) and may advantageously result in broader distribution of thevirus vector or capsid than would be observed in the absence of thepresent invention.

In particular embodiments, the delivery vectors of the invention may beadministered to treat diseases of the CNS, including genetic disorders,neurodegenerative disorders, psychiatric disorders and tumors.Illustrative diseases of the CNS include, but are not limited toAlzheimer's disease, Parkinson's disease, Huntington's disease, Canavandisease, Leigh's disease, Refsum disease, Tourette syndrome, primarylateral sclerosis, amyotrophic lateral sclerosis, progressive muscularatrophy, Pick's disease, muscular dystrophy, multiple sclerosis,myasthenia gravis, Binswanger's disease, trauma due to spinal cord orhead injury, Tay Sachs disease, Lesch-Nyan disease, epilepsy, cerebralinfarcts, psychiatric disorders including mood disorders (e.g.,depression, bipolar affective disorder, persistent affective disorder,secondary mood disorder), schizophrenia, drug dependency (e.g.,alcoholism and other substance dependencies), neuroses (e.g., anxiety,obsessional disorder, somatoform disorder, dissociative disorder, grief,post-partum depression), psychosis (e.g., hallucinations and delusions),dementia, paranoia, attention deficit disorder, psychosexual disorders,sleeping disorders, pain disorders, eating or weight disorders (e.g.,obesity, cachexia, anorexia nervosa, and bulemia) and cancers and tumors(e.g., pituitary tumors) of the CNS.

Disorders of the CNS include ophthalmic disorders involving the retina,posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabeticretinopathy and other retinal degenerative diseases, uveitis,age-related macular degeneration, glaucoma).

Most, if not all, ophthalmic diseases and disorders are associated withone or more of three types of indications: (1) angiogenesis, (2)inflammation, and (3) degeneration. The delivery vectors of the presentinvention can be employed to deliver anti-angiogenic factors;anti-inflammatory factors; factors that retard cell degeneration,promote cell sparing, or promote cell growth and combinations of theforegoing.

Diabetic retinopathy, for example, is characterized by angiogenesis.Diabetic retinopathy can be treated by delivering one or moreanti-angiogenic factors either intraocularly (e.g., in the vitreous) orperiocularly (e.g., in the sub-Tenon's region). One or more neurotrophicfactors may also be co-delivered, either intraocularly (e.g.,intravitreally or subretinally) or periocularly.

Uveitis involves inflammation. One or more anti-inflammatory factors canbe administered by intraocular (e.g., vitreous or anterior chamber)administration of a delivery vector of the invention.

Retinitis pigmentosa, by comparison, is characterized by retinaldegeneration. In representative embodiments, retinitis pigmentosa can betreated by intraocular (e.g., vitreal administration) of a deliveryvector encoding one or more neurotrophic factors.

Age-related macular degeneration involves both angiogenesis and retinaldegeneration. This disorder can be treated by administering theinventive delivery vectors encoding one or more neurotrophic factorsintraocularly (e.g., vitreous) and/or one or more anti-angiogenicfactors intraocularly or periocularly (e.g., in the sub-Tenon's region).

Glaucoma is characterized by increased ocular pressure and loss ofretinal ganglion cells. Treatments for glaucoma include administrationof one or more neuroprotective agents that protect cells fromexcitotoxic damage using the inventive delivery vectors. Such agentsinclude N-methyl-D-aspartate (NMDA) antagonists, cytokines, andneurotrophic factors, delivered intraocularly, optionallyintravitreally.

In other embodiments, the present invention may be used to treatseizures, e.g., to reduce the onset, incidence or severity of seizures.The efficacy of a therapeutic treatment for seizures can be assessed bybehavioral (e.g., shaking, ticks of the eye or mouth) and/orelectrographic means (most seizures have signature electrographicabnormalities). Thus, the invention can also be used to treat epilepsy,which is marked by multiple seizures over time.

In one representative embodiment, somatostatin (or an active fragmentthereof) is administered to the brain using a delivery vector of theinvention to treat a pituitary tumor. According to this embodiment, thedelivery vector encoding somatostatin (or an active fragment thereof) isadministered by microinfusion into the pituitary. Likewise, suchtreatment can be used to treat acromegaly (abnormal growth hormonesecretion from the pituitary). The nucleic acid (e.g., GenBank AccessionNo. J00306) and amino acid (e.g., GenBank Accession No. P01166; containsprocessed active peptides somatostatin-28 and somatostatin-14) sequencesof somatostatins as are known in the art.

In particular embodiments, the vector can comprise a secretory signal asdescribed in U.S. Pat. No. 7,071,172.

In representative embodiments of the invention, the virus vector and/orvirus capsid is administered to the CNS (e.g., to the brain or to theeye). The virus vector and/or capsid may be introduced into the spinalcord, brainstem (medulla oblongata, pons), midbrain (hypothalamus,thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland),cerebellum, telencephalon (corpus striatum, cerebrum including theoccipital, temporal, parietal and frontal lobes. cortex, basal ganglia,hippocampus and portaamygdala), limbic system, neocortex, corpusstriatum, cerebrum, and inferior colliculus. The virus vector and/orcapsid may also be administered to different regions of the eye such asthe retina, cornea and/or optic nerve.

The virus vector and/or capsid may be delivered into the cerebrospinalfluid (e.g., by lumbar puncture) for more disperse administration of thedelivery vector. The virus vector and/or capsid may further beadministered intravascularly to the CNS in situations in which theblood-brain barrier has been perturbed (e.g., brain tumor or cerebralinfarct).

The virus vector and/or capsid can be administered to the desiredregion(s) of the CNS by any route known in the art, including but notlimited to, intrathecal, intra-ocular, intracerebral, intraventricular,intravenous (e.g., in the presence of a sugar such as mannitol),intranasal, intra-aural, intra-ocular (e.g., intra-vitreous,sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon'sregion) delivery as well as intramuscular delivery with retrogradedelivery to motor neurons.

In particular embodiments, the virus vector and/or capsid isadministered in a liquid formulation by direct injection (e.g.,stereotactic injection) to the desired region or compartment in the CNS.In other embodiments, the virus vector and/or capsid may be provided bytopical application to the desired region or by intra-nasaladministration of an aerosol formulation. Administration to the eye, maybe by topical application of liquid droplets. As a further alternative,the virus vector and/or capsid may be administered as a solid,slow-release formulation (see, e.g., U.S. Pat. No. 7,201,898).

In yet additional embodiments, the virus vector can used for retrogradetransport to treat and/or prevent diseases and disorders involving motorneurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscularatrophy (SMA), etc.). For example, the virus vector can be delivered tomuscle tissue from which it can migrate into neurons.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

Example 1 Materials and Methods

Plasmids and Viruses.

All plasmids were obtained from the University of North Carolina GeneTherapy Center Vector Core facility. Virus was generated via tripletransfection of HEK293 cells and purified by cesium chloride densityultracentrifugation. Viral titer was obtained using real timequantitative PCR (qPCR) analysis, using the following primer setdesigned to recognize the luciferase transgene: (forward) 5′-AAA AGC ACTCTG ATT GAC AAA TAC-3′ (SEQ ID NO:9) and (reverse) 5′-CCT TCG CTT CAAAAA ATG GAA C-3′ (SEQ ID NO:10); or the following primer set designed torecognize human al anti-trypsin gene: (forward) 5′-GAA GTC AAG GAC ACCGAG GA-3′ (SEQ ID NO:11) and (reverse) 5′-CCC AGC TGG ACA GTC TCT TA-3′(SEQ ID NO:12). For all experimental groups, relevant viral constructswere titered together on the same qPCR plate prior to being used.Site-directed mutagenesis (Stratagene QuikChange) was used fornucleotide deletion or substitution. Table 8 lists the primer sequencesused for all VR1 mutants examined in this study. rAAV1→rAAV6 pointmutants (e.g., rAAV6.1, etc) were already part of general lab stocks.DNA sequencing was performed on each plasmid used in this work to verifysequence identity prior to making virus.

TABLE 8 Primers used for site-directed mutagenesis of VR1 mutants. NameSequence (listed 5′ to 3′) rAAV1/T265D gca atc tcc agt gct tca gac ggggcc agc aac g (SEQ ID NO: 13) rAAV1/T265E gca atc tcc agtngct tca gagggg gcc agc aac g (SEQ ID NO: 14) rAAV1/T265F gca atc tcc agtgct tca ttc ggg gcc agc aac gac (SEQ ID NO: 15) rAAV1/S261delcaa gca aat cag tgc ttc aac ggg gg (SEQ ID NO: 16) rAAV1/S262delgca aat ctc cgc ttc aac ggg gg (SEQ ID NO: 17) rAAV1/A263delgca aat ctc cag ttc aac ggg ggc (SEQ ID NO: 18) rAAV1/S264delcaa atc tcc agt gct acg ggg gcc aga aac (SEQ ID NO: 19) rAAV1/T265del*ctc cag tgc ttc agg ggc cag caa cg (SEQ ID NO: 20) rAAV1/G266delcca gtg ctt caa cgg cc gca agc (SEQ ID NO: 21) rAAV1/A267delgct tca acg ggg agc aac gac aac c (SEQ ID NO: 22) rAAV1/S268delcgg ggg cca acg aca acc ac (SEQ ID NO: 23) rAAV1/N269del cgg ggg cca gcgaca acc act tc (SEQ ID NO: 24) *exact rAAV1/T265del primer was used tocreate rAAV6/T265del mutants.

Live-Animal Studies.

All animals were maintained and treated in accordance with NationalInstitutes of Health guidelines, under protocols approved by the IACUCat the University of North Carolina, Chapel Hill. rAAV variantspackaging either the CBA-luciferase or CBA-hAAT transgene were injectedinto the GC of 6-8 week old female BALB/c mice (Jackson Laboratories) atindicated quantities. All injections were performed in 50 μL totalvolume per injection. Mice were anesthetized using isoflurane gas priorto injection. Luciferase transgene expression was monitored at indicatedtime points using a Xenogen IVIS Lumina imaging system (PerkinElmer/Caliper Life Sciences) following intraperitoneal injection ofD-luciferin substrate (Nanolight) at 120 mg/kg body weight.Bioluminescent image analysis was performed using Living Image software(Perkin Elmer/Caliper Life Sciences), and luciferase expression isreported in CPM/ROI (counts per minute over a selected region ofinterest).

Ex Vivo Quantitation of Transgene Expression and Copy Number.

For ex vivo luciferase assay, mice were sacrificed at 7 dpi of 1e10vgand injected muscle tissue harvested. Approximately 50 mg of each tissuewas minced on ice. Each tissue aliquot was then homogenized in 150 μL of2× Passive Lysis Buffer (Promega) using a Tissue Tearor (Cole-Parmer).35 μL of lysates and 100 μL of D-luciferin substrate (Promega) weretransferred to 96-well plates for luminometric analysis using a Victor2luminometer (Perkin Elmer). Total protein concentration in tissuelysates was determined via Bradford assay (Bio-Rad). At 3 dpi of 1e10vg,injected GC was harvested/homogenized and lysed as above, and a ˜25 mgtissue aliquot was processed using a DNeasy kit (Qiagen) to extract hostand vector genomic DNA. The number of cells was determined via qPCR withthe following primers designed specific to the mouse Lamin (ahousekeeping gene): (forward) 5′-GGA CCC AAG GAC TAC CTC AAG (SEQ IDNO:25) and (reverse) 5′-AGG GCA CCT CCA TCT CGG AAA C-3′ (SEQ ID NO:26).The number of viral genomes per cell was determined via qPCR. withprimers designed specific to the luciferase transgene, as describedabove.

Heparin Competition.

Indicated constructs were incubated in either a 250 μg/mL solution ofporcine heparin sulfate sodium salt (Sigma-Aldrich) dissolved inRinger's saline solution (RSS), or Ringer's saline solution alone,overnight at 4° C. with rotation. 1e10vg of each group was injected perGC in a total volume of 50 μL per injection. At 7 dpi, live animalbioluminescent imaging was performed.

Molecular Modeling.

The three-dimensional structures of rAAV1, rAAV2 or rAAV6 were displayedas whole capsids (a kind gift from Dr. Mavis Agbandje-McKenna) usingpreviously published coordinates in PyMol (pymol.org). The PyMol aligncommand was used to align various structures. Atom pairs involved inhydrogen bonding were calculated on crystal structure coordinates usingMolProbity and visualized using KiNG graphic software(kinemage.biochem.duke.edu).

Heparin Affinity Chromatography.

Indicated constructs were incubated with heparin sulfate conjugatedagarose beads (Sigma-Aldrich) overnight at 4° C. with rotation. Slurrywas transferred to a micro chromatography column (Bio-Rad) and washed 3times with RSS, followed by increasingly stringent NaCl washes.Solutions were made by dissolving appropriate quantity of NaCl into RSS,after adjusting for amount of NaCl already present in the solution. Allwash fractions were collected. Percent virus contained in each fractionwas determined by qPCR quantification of viral particles containedwithin each wash, using primers designed against the luciferasetransgene, described above.

ELISA Detection of Human AAT.

Mouse blood was collected via retro-orbital bleed using heparinizedcapillary tubes (Sigma-Aldrich). Immediately following collection,samples were pelleted and serum collected and stored at −80° C. forfuture use. Mice were anesthetized with isoflurane gas prior tobleeding. A 96-well plate was coated with rabbit anti-human AAT antibody(Sigma-Aldrich) at 10 mg/mL overnight at 4° C. Plate was then washed andblocked with serum dilution solution (PBS with 2.5% bovine albumin and0.05% Tween) for 1 hour at room temperature. Indicated serum dilutions(100 μL total per each sample) were added to plate and incubated for 2hours at room temperature. After washing 4 times with PBS, 100 μL ofHRP-conjugated goat anti-human AAT antibody (10 ug/mL; Abcam) was addedto plate for 1 hour at room temperature. After further washing, colorwas developed by addition of the TMB substrate (Pierce) and arrested by10% H₂SO₄. Optical density was read using an iMark microplate reader(Bio-Rad). Murine AAT was not detectable with this assay.

Example 2 Mutation of Position 265 on the rAAV1 Capsid EnhancesTransduction of Skeletal Muscle

As insertions of aspartic acid, glutamic acid or phenylalanine followingposition 264 in the rAAV2 capsid (creating a de novo position 265)enhanced skeletal muscle transduction by an order of magnitude, wehypothesized that substitution of the naturally present threonine atposition 265 in rAAV1 with D, E, or F would enhance skeletal muscletransduction in this serotype (rAAV1 is one amino acid longer than rAAV2in this capsid loop). As a control, T265 was deleted from rAAV1, so asto resemble unmodified rAAV2. rAAV1, rAAV1/T265D, rAAV1/T265E,rAAV1/T265F and rAAV1/T265del capsids packaging the chicken β-actinpromoter driven firefly luciferase (CBA-luc) transgene were injectedinto murine gastrocnemius (GC) muscle at a dose of 1e10 viral genomes(vg). Live-animal bioluminescent imaging was performed 7 days postinjection (dpi) in order to quantify transgene expression viameasurement of emitted light (FIG. 1A). rAAV1/T265D, rAAV1/T265E andrAAV1/T265F enhanced transgene expression relative to rAAV1 by 28-, 34-and 36-fold, respectively, supporting our previous analysis in the rAAV2capsid backbone. Surprisingly, rAAV1/T265del enhanced transduction byapproximately 200-fold over rAAV1. In an effort to better understandthis phenomena, the 265 deletion mutant was used in the majority ofremaining analyses within this study.

To confirm that the image quantification method used to generate theabove data was an accurate representation of transgene expression levelswithin injected muscle tissue, injected GCs were removed,homogenized/lysed and ex vivo luciferase assay performed on tissuelysate. In this assay, transduction efficiency with rAAV1/T265del wasenhanced by 137-fold over rAAV1, when normalized for relative lightunits emitted per mg of tissue analyzed (FIG. 1B). IncreasedrAAV1/T265del reporter gene expression correlated with enhancedtransgene delivery to muscle: 14.5-fold more viral genome copies weredetected per muscle cell relative to rAAV1 by quantitative real-time PCR(qPCR) analysis (FIG. 1C). All results were confirmed in multipleexperiments using independent preparations of vectors purified byseveral methods (e.g., density ultracentrifugation, affinitychromatography) to ensure that measured effects were not specific tobatch or purification method.

To ensure an appropriate time point was examined, a time course of rAAV1and rAAV1/T265del transgene expression was performed, with measurementscollected at 7, 14, 21 and 42 dpi (FIG. 1D). Expression kineticsappeared the same, with robust expression observed at 7 dpi, increasingby 7.5-fold between days 7 and 42 following injection of rAAV1 and by12-fold during same time period following injection of rAAV1/T265del. Atall time points, rAAV1/T265del outperformed rAAV1 by approximately twoorders of magnitude (100-fold at day 7, 176-fold at day 14, 81-fold atday 21, and 159-fold at day 42). Taken together, these data demonstratethat deletion of amino acid 265 in the rAAV1 capsid backbone is a highlyeffective strategy to enhance both transgene delivery and expression inskeletal muscle tissue. More importantly, this enhancement is maintainedover a sustained period of time supporting our observation of increasedvector genome copy numbers as documented by qPCR analysis.

Example 3 Distal Residues Work in Conjunction with 265 to ModulateTransduction Efficiency

rAAV6 is considered another top candidate for musculoskeletal genetherapy applications, and is the serotype with the highest capsidsequence homology (99.2%) to rAAV1. To determine whether the enhancedtransduction phenotype obtained by the deletion of position 265 in therAAV1 capsid would be conserved in the context of rAAV6, rAAV6 andrAAV6/T265del capsids packaging CBA-luc were generated. Seven daysfollowing injection of each into murine GC at a dose of 1e10vg,transgene expression was quantified. Intriguingly, the transgeneexpression following injection of rAAV6/T265del was only 82% of thatmeasured when using rAAV6 (FIG. 2A). This was surprising considering thedramatic effects on transduction that deletion of position 265 producedin rAAV1, and that of the only 6 amino acids by which rAAV1 and rAAV6differ, none are located within sufficient spatial proximity to position265 to form direct interactions. Furthermore, any residue that differsbetween rAAV1 and rAAV6 is located at least 22 Å away from position 265.In order to form direct interactions such as hydrogen bonding, residuesmust be within ˜3 Å of each other. Despite this, there areconformational differences within the structure of rAAV1 and rAAV6around position 265 (FIG. 2B), as visualized by alignment of availablecrystallographic data for each.

To resolve which residues within the rAAV6 capsid might inhibit theeffectiveness that deleting position 265 has on enhancing transductionefficiency a version of marker rescue experiment was employed, whereinthe rAAV6 capsid was mutated at each residue that differs between rAAV1and rAAV6 to the amino acid present in rAAV1, in conjunction withdeletion of 265. Mutants were designated rAAV6.1 (rAAV6 with the firstnon-conserved residue (F129L) mutated to the equivalent amino acid inrAAV1), rAAV6.2 (D418E), rAAV6.3 (K531E), rAAV6.4 (L584F), rAAV6.5(V598A) and rAAV6.6 (H642N), respectively. The majority of changes tothe rAAV6 capsid produced minimal change to transduction (FIG. 2C).Transgene expression decreased by ≤2-fold with constructs rAAV6.1,rAAV6.1/T265del, rAAV6.2, rAAV6.2/T265del and rAAV6.3, respectively.Transgene expression was increased by approximately 2-fold in bothrAAV6.4 and rAAV6.4/T265del. Mutations at position 598 and 642 (rAAV6.5and rAAV6.6) reduced transgene expression relative to rAAV6 moredramatically than other mutations, decreasing it by 3.4- and 13.1-fold,respectively. Deletion of position 265 in conjunction with rAAV6.5 andrAAV6.6 produced little change to the performance of these constructs,decreasing transgene expression by 4.8- and 8.7-fold in rAAV6.4/T265deland rAAV6.5/T265del, respectively. Strikingly, only a single amino acidchange, at position 531 (rAAV6.3), was required to rescue the 265phenotype observed with rAAV1, with rAAV6.3/T265del enhancingtransduction efficiency by 23-fold relative to rAAV6 (FIG. 2C).

The spatial relationship between residues 531 and 265 was examined inthe context of the rAAV6 crystal structure (FIGS. 3A-3D). Amino acids265 and 531 are not close enough to form direct interactions in any ofthe icosahedral axes of symmetry. In the 2-fold axis, positions 265 and531 are ˜26.7 Å apart, whereas in the 5-fold axis, they are ˜42.6 Åapart. However, in the 3-fold axis of symmetry, despite being located˜22.8 Å apart, both residues are surface-exposed in the same plane (FIG.3B). Residue 531 sits at the base of each 3-fold “spike” and 265 sits ina slight protrusion between spikes. Taken together, at this point intime, the above data suggests that the most likely context in whichpositions 265 and 531 may together modulate transduction efficiency isthrough the protrusions forming the 3-fold axis of symmetry.

Example 4 Capsid Heparin Binding Ability Affects the 265 Phenotype, andVice-Versa

rAAV1 and rAAV6 both engage N-linked sialic acid as a primary receptor;however, rAAV6 also binds to heparin sulfate moieties. Previously, ourresults have shown that K531 is the sole residue required for rAAV6 tointeract with heparin sulfate. K531 is also the sole residue required torescue the 265 deletion phenotype observed in rAAV1. To further explorethe functional relationship between capsid heparin binding and theenhanced transduction resulting from mutation of position 265 weexamined rAAV2, the prototypical serotype known to utilize heparinsulfate proteoglycans as primary receptors for infection. rAAV2 wasmutated at position 585 (a mutation known to attenuate rAAV2 heparinbinding) in conjunction with insertion of aspartic acid to create a denovo position 265 as previously described. rAAV2, rAAV2/265D,rAAV2/K585E and rAAV2/265D,K585E were injected into murine GC at a doseof 1e10vg. In agreement with previous reports, ablation of heparinbinding enhanced rAAV2 transduction of skeletal muscle. However,concomitant mutation of position 265 and elimination of heparin bindingability produced the strongest enhancement of transduction efficiency(FIG. 4A), increasing transduction by 96-fold over rAAV2 and 7-fold overrAAV2/265D and in agreement with above results observed for rAAV6 (FIG.2C). It is notable that residue 531 was mutated to ablate heparinbinding in rAAV6 and residue 585 was mutated to ablate heparin bindingin rAAV2. Though residues 531 and 585 are not close to each other inlinear sequence, they both fall within an overlapping basic patch on thecapsid surface that is the footprint for heparin binding (FIG. 4B).

To resolve whether direct inhibition of heparin binding enhancestransduction of 265 mutants, heparin competition was performed prior toinjection of 265 mutant constructs into mice. rAAV2 binds heparin withstronger affinity than rAAV6, and is the only serotype historicallyshown to have effective diminution of transduction following incubationin a heparin sulfate solution, so this question was examined in thecontext of rAAV2. rAAV2 and rAAV2/265D capsids were incubated in eithera saline solution or a 250 μg/mL heparin sulfate solution overnight.rAAV1 was included as a control. Viruses were injected into murine GC ata dose of 1e10vg, and mice were imaged 7 dpi. Incubation with heparinsulfate reduced transduction efficiency by approximately 3-fold in bothrAAV2 and rAAV2/265D and had virtually no effect on rAAV1, reducingtransduction by 0.3-fold (FIG. 4C). These results suggest that the needto ablate heparin sulfate binding in order for mutation of position 265to be effective at enhancing transduction is due to structural changewithin the capsid and not inhibition of heparin binding, per se.

Heparin affinity chromatography verified that all rAAV2 and rAAV6 capsidmutations made to decrease heparin binding performed as expected (FIGS.5A-5D). Relevant constructs were incubated with heparin-conjugatedagarose and eluted with an increasing NaCl gradient. Interestingly, incapsids with position 265 mutated, heparin binding was attenuatedrelative to parental capsids. For example, 20% more rAAV2/265D thanrAAV2 eluted during the first wash step following column loading ofvirus. Similarly, 20% less rAAV2/265D eluted in the 300 mM NaCl fractionthan did rAAV2. Likewise, 37% more rAAV6/T265del eluted during columnwashing than rAAV6, while 25% less rAAV6/T265del eluted at the 200 mMNaCl wash than did rAAV6, supporting the hypothesis of interplay betweenthese two protein motifs (elution of rAAV6 during a less stringent washfraction than rAAV2 agrees with previous work). While rAAV2/K585Eattenuated rAAV2 heparin binding, rAAV2/265D,K585E furthered thiseffect. 75% of rAAV2/K585E and 96.5% of rAAV2/265D,K585E was elutedduring initial wash steps. 10% less rAAV2/265D,K585E than rAAV2/K585Ewas eluted at 200 mM NaCl. Combined with the data presented in FIGS. 3and 4, these results add evidence that changes to the capsid regionharboring position 265 affect the structural conformation of that regionharboring the heparin binding site, and vice-versa.

Example 5 Multiple Disruptions to VR1 Structure Enhance Skeletal MuscleTransduction

The data presented thus far suggests that changes in the structuralconformation of VR1 alone is the driving force behind the enhancedtransduction following mutation of position 265. Therefore, we sought todetermine whether additional disruption to VR1 structure could produce asimilar enhanced transduction phenotype. rAAV1 was thus mutated togenerate S261del, S262del, A263del, S264del, G266del, A267del, S268deland N269del, as illustrated in FIG. 6A. These constructs were injectedinto murine GC at 1e10vg, alongside rAAV1 and rAAV1/T265del. Transgeneexpression was quantified 7 dpi (FIG. 6B). Each mutant enhancedtransduction efficiency to varying degrees relative to rAAV1.Transduction was enhanced by ≤3-fold in the S261, S262 and N269 deletionconstructs. The G266 and S268 deletion constructs enhanced transductionby ˜5-fold and the A263del and A267del by ˜10-fold relative to rAAV1.The S264 and T265 deletion constructs enhanced transduction the mostrobustly over rAAV1, increasing transgene expression by 25- and100-fold, respectively. These data indicate that any disruption made tothe VR1 loop via deletion of an amino acid will enhance transduction inrAAV1, with such enhancement peaking via deletion of position 265 andtapering off as mutations are made further away from position 265 (FIG.6B).

Example 6 265 Deletion Mutants Improve Expression of a TherapeuticTransgene

To compare the relative efficacy of the 265-deletion panel, six capsidswere packaged with the hAAT expression cassette currently in clinicaltrial use. rAAV1, rAAV1/T265del, rAAV6, rAAV6/T265del, rAAV6.3 andrAAV6.3/T265del capsids were injected into murine GC at 1e10vg. Serumconcentrations of hAAT were measured via ELISA 5 weeks post injection(FIG. 7). In agreement with above, transgene expression was enhanced bydeletion of position 265; however, the degree of enhancement did notreach the levels measured by luciferase assay (e.g., by ELISA there wasa 9-fold increase in rAAV1/T265del expression over rAAV1, whereas theaverage measured by luciferase assay was >100-fold). Nonetheless, all265 mutants outperformed parent serotypes, with rAAV6.3/T265delproducing the greatest serum concentration of hAAT.

Example 7 Enhanced Transduction Efficiency in the Eye

AAV constructs were injected either subretinally (SRI) or intravitreally(IV) into mice anesthetized with a ketamine/xylazine cocktail. All AAVcapsids packaged the reporter gene luciferase under the chicken betaactin promoter (CBh). Constructs that were examined included AAV1(1-CBh), AAV1/T265del (1.1-CBh), AAV6/T265del (6.1-CBh), AAV6/K531E(6.3-CBh), AAV6/T265del_K531E (6.3.1-CBh), and an AAV2 variantpreviously characterized in our lab, AAV2.5 (2.5-CBh) (Bowles et al.,Mol. Ther. 20(2):443 (2012)). Transgene expression was measured asluciferase activity at indicated time points post injection, using theIVIS lumina live animal imaging system. Mice were imaged followingintraperitoneal injection of D-luciferin substrate (Nanolight) at 120mg/kg body weight, and images were quantified by measuring photonswithin a given region of interest, according to IVIS manufacturerinstructions. Results are shown in FIGS. 9 and 10.

Success in the translation of rAAV gene therapy has been challenged bylow transduction efficiency in vivo. This study employed structuralanalysis of the rAAV capsid to develop a rational engineering strategythat can clearly yield novel capsid variants with enhanced transductionphenotypes in skeletal muscle. The overall approach was made possible byearlier reports from our lab, which uncovered VR1 as a key determinantof transduction efficiency in rAAV2. As we had previously discovered inrAAV2, enhancement of transduction was observed with substations ofvarious amino acids at position 265 in rAAV1. Remarkably, however,disruption of VR1 via the deletion of single amino acids in the moreclinically relevant rAAV1 yielded novel vectors with transductionenhanced by orders of magnitude (FIG. 1). This observation alone hassignificant implications for patient dosing protocols currentlyutilizing rAAV1 and may lead to vector production easement movingforward. It was noted that in the rAAV6 capsid a similar outcome wasobtained, but only after secondary mutations was created to disrupt thebasic amino acid patch previously shown to confer heparin sulfatebinding activity. Using a marker rescue approach made possible byanalyzing capsid sequence homology, two distal regions of the capsidsurface were defined that work in concert to modulate transductionefficiency and primary receptor recognition, shedding light on thecomplex role of capsid topology during the viral life-cycle.

The antagonistic relationship between position 265 and primary receptorbinding suggests that the 265 deletion phenotype results from amodification in protein:protein interactions between the capsid and abinding partner. Whether this occurs at initial attachment to the cellsurface or during a post entry event remains to be determined. That theonly measurable correlation to transgene expression was an increase inthe number of vector genomes per cell, and not in expression kinetics orlongevity, strongly suggests that the 265 phenotype is a result ofalterations in cellular entry phenomena. VR1 has been implicated inreceptor binding in rAAV2, as well as in a chimeric vector generated viadirected evolution, though explicit structural details of the nature ofthese interactions have not yet been defined. The spatial relationshipof position 265 relative to the protrusions that comprise the three-foldaxis of symmetry—the primary region of the capsid responsible forreceptor engagement—lends further evidence to this idea. Of note, nodifferences were seen between wild-type and mutant constructs in 1.)heat denaturation assays testing capsid stability, 2.) Western blottingto examine the ratio and size of capsid proteins, or 3.) in electronmicroscopy to search for overt differences in capsid topology orpopulation distribution of empty versus full capsids. Furthermore, doseresponse data suggest that the 265 enhanced transduction profile can bedialed in for respective clinical applications (e.g., AAT vs lipoproteinlipase deficiency).

It is well known that phosphorylation of threonine residues effectsintracellular protein dynamics. Indeed, the phosphorylation of selectserine, threonine and tyrosine residues on the rAAV capsid surfaceultimately results in proteasomal clearance of capsids prior totransgene delivery and mutation of these residues to non-phosphorylablespecies prevents capsid clearance, thereby enhancing transductionefficiency. That multiple different types of amino acid at position 265(deletion of threonine, substitution with phosphomimetics or bulkyhydrophobics) all enhanced transduction suggests that phosphorylation isnot likely responsible for transduction enhancements mediated by 265mutation. Furthermore, the structure of VR1 could be disruptedthroughout the loop to obtain an improved transduction phenotype. It ispossible that these disruptions relieve the capsid from interacting withan inhibitory protein, though this has yet to be determined. However,there is evidence of a similar effect in rAAV2, in which ablation ofcapsid heparin binding ability substantially improves transduction ofmuscle tissue^(((null)(null))). Indeed, such data was recapitulated inthis work. It has been argued that the ability of rAAV2 to bind heparinsulfate proteoglycans has evolved through tissue culture adaptation ofthe virus and that binding to such receptors has been detrimental to thein vivo transduction efficiency of this serotype. Likewise, in the caseof adenovirus, robust hepatic transduction is maintained even when thecapsid is mutated to exclude binding to its primary tethering receptor,CAR, as alternate regions of the capsid are able to compensate byfacilitating productive interactions with alternate receptors.

It is interesting that transduction efficiency peaked following deletionof position 265, specifically. In analyzing the hydrogen bonding networkof VR1 in rAAV1 (FIG. 8A), it is noteworthy that VR1 is stabilized by anetwork of hydrogen bonds primarily orchestrated by position 265.Indeed, the next best transducers resulted from deletion of positions264 and 263, both of which also participate in the structuralstabilization of VR1 via this network. Of course, the only way to trulyexamine the effects of these mutations on the stability of VR1 would bethrough crystallographic resolution of these constructs. It issignificant, however, that rAAV6 is inherently less stabilized aroundposition 265 (FIG. 8B), and that in practice rAAV6 requires additionalmutation to replicate the position 265 phenotype. Many studies haveattempted to unravel the phenotypic disparity (e.g., differences inreceptor preferences and tissue tropism) between the closely relatedrAAV1 and rAAV6. Virtually all previous studies have examined the sixcapsid amino acids that differ between these two viruses, with theunderstanding that pinpointing these locations allows dissection ofcapsid regions that directly influence the viral life-cycle. While thisis true, we demonstrate here for the first time an additional, far moresubtle layer of capsid architecture that differs on a structural levelbetween these two serotypes to regulate viral biology, along with arational design approach to exploit these attributes.

Another compelling finding is the discrepancy of measured transductionenhancement between rAAV1 and rAAV1/T265del when examining transgeneexpression within injected tissue directly (i.e., ex vivo luciferaseassay, which produced 137-fold enhancement) versus measurement oftransgene expression of a secreted protein (i.e., ELISA to determine theconcentration of hAAT, which produced 9-fold enhancement). It isnoteworthy that in previous experiments comparing the measurements ofsecreted hAAT from i.m. injected rAAV2, rAAV2.5 and AAV2/265D, noobvious differences were found by ELISA, despite order of magnitudeincreases in luciferase expression relative to rAAV2 when using theengineered capsids. Likewise, while it has been found that theproteasome inhibitor Bortezomib can enhance rAAV2 transduction in livertissue by approximately 10-fold when measured by luciferase assay, thatenhancement drops to less than 2-fold when measuring serum for theexpression of secreted Factor IX. It is likely that measuring transgeneexpression directly from harvested tissue is a more sensitive techniquethan is the collection and subsequent measurement of secreted factorsdue to differences such as protein half-life within an intracellularenvironment versus serum, as well as overall efficiency of proteinsecretion relative to protein expression. Such findings highlight theexquisite increase in transduction that needs to be achieved fortranslatable applicability when designing rAAV vectors meant to delivertransgenes encoding secreted proteins.

The goal of these efforts is the continued improvement of rAAV vectortechnology so that gene therapy becomes a more practical and effectiveclinical choice, such as through reducing vector dosing required toachieve therapeutically beneficial transgene levels. Such efforts mayallay clinical concerns around vector dose-dependent toxicity and easeclinical-grade vector production efforts. Here we present a simple andeffective strategy for improving the performance of rAAV serotypescurrently being used in clinical trials targeting skeletal muscle bydirect injection. As this strategy utilizes a region of the capsidunique to other major advancements in rAAV vector development (e.g., theTyr-to-Phe capsid mutants), it will be interesting to determine whetherit can be integrated synergistically with such technologicaladvancements to further optimize rAAV vectors for gene therapy. As wecontinue evaluating the potential of our 265 mutant panel to target andenhance transgene expression in cardiac tissue, as well as whole bodyskeletal muscle following systemic administration, the work containedherein provides a starting foundation for the development of nextgeneration translational rAAV vectors. Furthermore, we have produced acollection of rAAV capsid reagents available for further detailed (e.g.,crystallographic) analysis that will eventually lead to a morecomprehensive understanding of the unique and significant role thatsingle amino acids play in rAAV vector biology.

Example 8 Materials and Methods

Plasmids and Viruses:

All plasmids were obtained from the University of North Carolina GeneTherapy Center Vector Core facility. Virus was made by way of tripletransfection of HEK293 cells and purified by cesium chloride densityultracentrifugation, as previously described (Grieger et al., Nat.Protoc. 1(3):1412 (2006)). Viral titer was measured using real timequantitative PCR (qPCR) analysis, with the following primer set designedto recognize the luciferase transgene: (forward) 5′-AAA AGC ACT CTG ATTGAC AAA TAC-3′ (SEQ ID NO:10) and (reverse) 5′-CCT TCG CTT CAA AAA ATGGAA C-3′ (SEQ ID NO:11). For each experimental group, relevant viralconstructs were titered together on the same qPCR plate prior to beingused. Site-directed mutagenesis (Stratagene QuikChange) was used tocreate nucleotide deletions or substitutions. Table 9 lists the primersequences used for all VR1 mutants created in this study. Prior tomaking virus, DNA sequencing was performed to verify that the sequenceidentity of each plasmid used in this work was correct.

TABLE 9 Site-directed mutagenesis primers used in this study. ConstructPrimer Sequence (5′ → 3′) pXR1/T265del*ctc cag tgc ttc agg ggc cag caa cg (SEQ ID NO: 27) pXR1/T265D*gca atc tcc agt gct tca gac ggg gcc agc aac g (SEQ ID NO: 13)pXR1/Y445F* cat cga cca gta cct gta ttt cct gaacag aac tca (SEQ ID NO: 28) pXR2/Q263delcaa aca aat ttc cag ctc agg agc ctc g (SEQ ID NO: 29) pXR2/S264delcaa att tcc agc caa gga gcc tcg aac g (SEQ ID NO: 3026) pXR3b/Q263delcaa gca aat ctc cag ctc agg agc ttc (SEQ ID NO: 31) pXR3b/266Dctc cag cca atc aga cgg agc ttc aaa cg (SEQ ID NO: 32) pXR3b/R594Acag ctc cca cga ctg caa ctg tca atg atc (SEQ ID NO: 33) pXR4/N261delgag cct gca gtc cac cta caa cgg (SEQ ID NO: 34) pXR5/258delccg tcg acg gaa acg cca acg cc (SEQ ID NO: 35) pXR6/S262delgca aat ctc cgc ttc aac ggg gg (SEQ ID NO: 36) pXR7/T265delcaa atc tcc agt gaa gca ggt agt acc (SEQ ID NO: 37) pXR8/T265delctc caa cgg gtc ggg agg agc (SEQ ID NO: 38) pXR8/T265Dcaa atc tcc aac ggg gac tcg gga gga gcc acc (SEQ ID NO: 39) pXR8/S266deltct cca acg gga cag gag gag cca acc (SEQ ID NO: 40) pXR9/S263delcaa gca aat ctc cag cac atc tgg agg (SEQ ID NO: 41) pXR9/S265delctc caa cag cac agg agg atc ttc aaa tg (SEQ ID NO: 42) pXR9/T265Dcaa gca aat ctc caa cag cga ctc tgg agg atc ttc aaa tg (SEQ ID NO: 43)pXR9/S263A_ gca aat ctc caa cgc cac agg agg atc S266delttc aaa tga c (SEQ ID NO: 44) *indicates that same primer sequence wasused for pXR1 and pXR6 constructs

Structural Analysis and Molecular Modeling:

Indicated pdb files were processed through MolProbity structuralvalidation software (molprobity.biochem.duke.edu) to add hydrogen bondsbased on electron-cloud x-H bond lengths, using H-bond optimization viathe inclusion of Asn/Gln/His flips where necessary. Files were thenanalyzed for all-atom contacts and geometry, and visualized in KiNG(kinemage.biochem.duke.edu) to determine VR1 atoms participating inhydrogen bonds. PDBs were then opened in PyMol (pymol.org), shown asstick diagrams with hydrogens visualized, and hydrogen bonds identifiedin KiNG were translated into dashed lines using the measure.

Live-Animal Studies:

Animals were maintained and handled in accordance with NationalInstitutes of Health guidelines, under protocols approved by the IACUCat the University of North Carolina, Chapel Hill. rAAV variantspackaging the CBA-luciferase transgene were injected into the tail veinof 6-8 week old female BALB/c mice (Jackson Laboratories) at indicatedquantities. All injections were performed in 200 μL total volume perinjection (volume was brought to 200 μL using 1×PBS). Luciferasetransgene expression was visualized using a Xenogen IVIS Lumina imagingsystem (Perkin Elmer/Caliper Life Sciences) following intraperitonealinjection of D-luciferin substrate (Nanolight) at 120 mg/kg body weight.Bioluminescent image analysis was performed using Living Image software(Perkin Elmer/Caliper Life Sciences).

Ex Vivo Quantification of Transgene Expression and Copy Number:

To obtain tissue samples for analysis via ex vivo luciferase assay, micewere sacrificed at 10 dpi and indicated organs were harvested and flashfrozen. Tissue was homogenized on ice using a razor blade, andapproximately 50 mg of each tissue homogenate was then suspended in 250μL of 2× Passive Lysis Buffer (Promega) and lysed mechanically using aTissue Tearor (Cole-Parmer). 35 μL of each tissue lysate was thentransferred to a 96-well plate and mixed with 100 μL of D-luciferinsubstrate (Promega) immediately prior to performing luminometricanalysis using a Victor2 luminometer (Perkin Elmer). The total proteinconcentration of each tissue lysate was determined by Bradford assay(Bio-Rad). Additionally, a ˜25 mg aliquot of the above generated tissuehomogenate was processed using a DNeasy kit (Qiagen) to extract host andvector genomic DNA. The number of cells was determined using qPCR withthe following primers designed specific to mouse Lamin (a housekeepinggene): (forward) 5′-GGA CCC AAG GAC TAC CTC AAG GG-3′ (SEQ ID NO:45) and(reverse) 5′-AGG GCA CCT CCA TCT CGG AAA C-3′ (SEQ ID NO:46), The numberof viral genomes per cell was also determined using qPCR with primersdesigned specific to the luciferase transgene, as described above.

Example 9 Structural Analysis of the rAAV Capsid to Identify HydrogenBond Networks in VR1

We have previously demonstrated that the deletion of select amino acidswithin VR1 of the rAAV1 capsid leads to log order increases intransduction efficiency following intramuscular injection into mice(Warischalk et al., Mol. Ther. 2015). Analysis of the rAAV1 crystalstructure revealed that the most efficient VR1 deletion mutationscorresponded to amino acids participating in intra-loop hydrogen bondnetworks, suggesting that the destabilization of VR1 structural elementsleads to enhanced transduction phenotypes. The goal of the present studywas to determine: 1) whether rAAV1 VR1 mutant capsids can efficientlytarget muscle tissue following intravenous injection (versus simplyhaving a phenotype of enhanced transduction), and 2) whether thetargeted destabilization of VR1 structural elements is a conservedmethod of improving transduction in additional rAAV serotypes. Toaddress these questions, a collection of rAAV capsids including rAAV1,rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, and rAAV9 were analyzedwith the open source structural validation software MolProbity (Davis etal., Nucleic Acids Res. 35 (Web Server issue):W375 (2007)) and VR1hydrogen bond patterns visualized in KiNG (Chen et al., Protein Sci.18(11):2403 (2009)) (FIGS. 11A-11I). These serotypes were chosen foranalysis based on the availability of both capsid crystal structure data(Govindasamy et al., J. Virol. 80(23):11556 (2006); Miller et al., ActaCrystallogr. Sect. F Struct. Biol. Cryst. Commun. 62(Pt 12):1271 (2006);Xie et al., Proc. Natl. Acad. Sci. USA, 99(16):10405 (2002); Lerch etal., Virology 403(1):26 (2010); Govindasamy et al., J. Virol.87(20):11187 (2013); Xie et al., Acta Crystallogr. Sect. F Struct. Biol.Cryst. Commun. 64(Pt 11):1074 (2008); Nam et al., J. Virol. 81(22):12260(2007); Mitchell et al., Acta Crystallogr. Sect. F Struct. Biol. Cryst.Commun. 65(Pt 7):715 (2009)) and pre-existing knowledge of their in vivotransduction phenotypes (Zincarelli et al., Mol. Ther. 16(6):1073(2008)). There is no publicly available structural information forrAAV7; however, this serotype was included for the sake of completeness.For ease of visualization, identified hydrogen bond patterns were thentranslated into PyMol images (FIGS. 12A-12H).

Based on these analyses, amino acids from each serotype that were foundto participate in intra-loop VR1 hydrogen bond networks were selectedfor deletion. Table 10 lists the VR1 amino acid sequence for serotypesrAAV1-rAAV9 and the amino acids chosen for mutation in this study, aswell as how many hydrogen bonds are hypothesized to be broken with eachmutation. In the case of rAAV7, mutation of the threonine at position265 was chosen based on previous results obtained for rAAV1 (Warischalket al., Mol. Ther. 2015). In the case of rAAV9, the structure of VR1appears to be stabilized by hydrogen bonds in two locations, the firstprimarily orchestrated by bonds originating around residue S263, and thesecond by bonds originating around residue S265. Therefore, mutantcapsids comprising rAAV9/S263del and rAAV9/S265del were created, inaddition to the double mutant rAAV9/S263del_S265del. However, the doubledeletion mutant was unable to produce intact viral particles, which ledto the alternate strategy of mutating position S263 to an alanine inconjunction with deletion of position S265, to createrAAV9/S263A_S265del. For detailed atomic descriptions of the hydrogenbond relationships observed for all of the serotypes included withinthis study, refer to the legend for FIG. 11.

TABLE 10 Variable region 1 amino acid sequencesand deletion mutations made in rAAV1-rAAV9. Serotype Variable Region 1Mutations rAAV1 ₂₆₁SSASTGASNDNHY₂₇₃ T265del+++ (SEQ ID NO: 1) rAAV2₂₆₁SSQSGAS₂₆₇ Q263del+, S264del+ (SEQ ID NO: 2) rAAV3b₂₆₁SSQSGASNDNHY₂₇₂ Q263del++ (SEQ ID NO: 3) rAAV4 ₂₅₆ESLQSNTY₂₆₃N261del+ (SEQ ID NO: 4) rAAV5 ₂₅₂SGSVDGS₂₅₈ S258del++ (SEQ ID NO: 5)rAAV6 ₂₆₁SSASTGASNDNHY₂₇₃ S262del++, T265del+ (SEQ ID NO: 6) rAAV7 n/aT265del rAAV8 ₂₆₃NGTSGGAT₂₇₀ T265del+ (SEQ ID NO: 7) rAAV9₂₆₁SNSTSGGSS₂₆₉ S263del++, S265del+, (SEQ ID NO: 8) S263del_S265del++++ indicates number of hydrogen bonds hypothesized to be broken followingmutation

Example 10 Biodistribution of rAAV Capsids Mutated to DestabilizeHydrogen Bond Networks in VR1

To visualize tissue distribution and transduction efficiency of theabove detailed wild-type and VR1 mutant capsids, each member of thecollection was packaged with the chicken β-actin promoter driven fireflyluciferase (CBA-luc) reporter transgene and injected into mice via thetail vein at a dose of 1e11 viral genomes (vg) per injection.Live-animal bioluminescent imaging was performed at 9 days postinjection (dpi). At 10 dpi, mice were sacrificed and select organsremoved, homogenized and lysed. Vector biodistribution was thenquantified by subjecting tissue lysates to ex vivo luciferase assays inorder to measure transgene expression, and qPCR analysis to quantifyviral genome copy numbers per cell. The deletion of VR1 amino acidsresulted in four phenotypes: 1) selective and robust targeting ofcardiac and/or skeletal muscle, as observed for rAAV1 and rAAV6 (FIG.13); 2) maintenance of innate cardiac and/or skeletal muscletransduction efficiency, coupled with a substantial reduction intransduction of the liver, as seen in rAAV7, rAAV8, and rAAV9 (FIG. 14);3) a widespread absence of transduction, as in rAAV2 and rAAV3 (FIGS.15A-15B); and, 4) the inability to produce intact viral particles, as inthe case of rAAV4 and rAAV5.

VR1 mutations in rAAV1 and rAAV6 capsids led to substantial gains intransduction efficiency in cardiac and skeletal muscle tissues (FIG.13). Transgene expression measured 21-fold higher in cardiac and 26-foldhigher in gastrocnemius (GC) tissue samples taken from mice treated withrAAV1/T265del versus rAAV1. rAAV1/T265del transduction efficiency wasalso enhanced in several other tissue types, including the diaphragm(26-fold), the spleen (3.5 fold), the kidney (5-fold), the pancreas(6-fold), and the lung (8-fold), (FIG. 16A). Surprisingly, there was a3.5-fold decrease in hepatic transduction relative to rAAV1 (FIG. 13B).Similarly, rAAV6/T265del capsids transduced cardiac and GC muscle atlevels 14-fold and 3-fold higher then wild-type rAAV6, concomitant witha 28-fold decrease in transduction of the liver (FIG. 13E). There wereno remarkable differences in measured transgene expression between rAAV6and rAAV6/T265del capsids in any other tissue tested (FIG. 16B).rAAV6/S262del produced a similar phenotype to rAAV6/T265del, transducingat levels 3.5-fold lower and 6-fold higher then rAAV6/T265del in cardiacand GC muscle, respectively, and within 1-fold of rAAV6/T265del in theliver (FIG. 16C). These results suggest that rAAV1 and rAAV6 VR1 mutantconstructs preferentially transduce muscle tissue when systemicallyadministered to mice.

The numbers of vector genomes present per host cell were quantified viaqPCR on a subset of organs including the heart, GC, and liver (FIGS.13C, 13F). In tissue samples obtained from mice treated withrAAV1/T265del and rAAV6/T265del capsids, changes in host cell transgenepopulations measured by qPCR trended similarly to those observed fortransgene expression measured by luciferase assay (i.e., more vectorgenomes correlated with increased transgene expression, and vice-versa).However, differences amongst transgene population measurements were moresubtle than those obtained for transduction efficiency. For example, inmice treated with rAAV6/T265del there were 4-fold more vector genomesper heart cell than in mice treated with rAAV6, whereas there was a14-fold increase in cardiac transduction with the mutant capsid (FIG.13F). Likewise, there were 4-fold fewer rAAV6/T265del genomes per livercell, versus a 28-fold decrease in transduction. Intriguingly, vectorgenomes per cardiac cell were substantially lower than those measuredper liver cell in the case of both rAAV1 and rAAV1/T265del, despite thattransduction of the heart was far greater than that of the liver in bothconstructs. Collectively, these data indicate that the reduced abilityof rAAV1 and rAAV6 VR1 mutant capsids to transduce the liver correspondswith an inability of these capsids to deliver a persistent transgene tohepatic cells. Additionally, transgenes delivered by VR1 mutant capsidsto muscle tissues appear to be more effectively expressed than thosedelivered by their wild-type counterparts. Finally, it would appear thatsome facet of the liver's microenvironment imposes a hurdle towards therAAV1 capsid's ability to either enter the nucleus or uncoat once there.

Deleting VR1 amino acids from rAAV7, rAAV8 and rAAV9 capsids createdbiodistribution profiles with strikingly similar phenotypes (FIGS. 14A,14B, 14G). The primary feature observed for each was a sharp reductionin hepatic transduction (427-fold in rAAV7, 23-fold in rAAV8 and107-fold in rAAV9 relative to wild-type capsids), while native cardiacand skeletal muscle transduction levels were maintained. Transductionefficiency in muscle tissue did not markedly change between rAAV7 andrAAV7/T265del; however, rAAV8/T265del enhanced transduction efficiencyin GC tissue by approximately 8-fold relative to rAAV8 (FIG. 14E).rAAV9/S263del_S265del produced variable effects in muscle tissue,transducing cardiac tissue 3-fold lower and GC tissue 2-fold higher thanrAAV9 (FIG. 14H). There were no prominent transduction differencesbetween VR1 mutant and wild-type capsids of this subset in any othertissue type sampled (FIGS. 17A-17C). It is notable that simultaneousmutations at positions S263 and S265 were required to produce amuscle-targeted biodistribution profile in rAAV9, and that singledeletions at either of these positions substantially reduced overalltransduction efficiency (FIG. 14D).

The numbers of vector genome copies present per host cells werequantified in cardiac, GC and hepatic tissue samples. In all VR1 mutantcapsids of this subset, reduced hepatic transgene expressioncorresponded to reduced transgene populations within liver cells(85-fold fewer vector genomes per liver cell for rAAV7/T265del, 27-foldfewer for rAAV8/T265del and 7-fold fewer for rAAV9/S263del_S265del;FIGS. 14C, 14F, 14I). The relationship between transgene population andtransduction efficiency was less clear in muscle tissue samples. Forexample, there was a 22-fold reduction in transgene copies per cardiaccell when comparing rAAV7/T265del to rAAV7, yet transduction efficiencybetween the capsids was virtually the same. These data suggest that VR1mutant capsids of this subset are deficient in the ability to deliver astable transgene population to hepatic cells, while transgenes deliveredby these capsids to muscle tissues are more effectively expressed thanthose delivered by their wild-type counterparts.

The deletion of VR1 amino acids produced deleterious effects in rAAV2,rAAV3b, rAAV4 and rAAV5 capsids. In rAAV4 and rAAV5, VR1 amino aciddeletions led to an inability to produce intact virions. Vectorproduction was attempted several times with both constructs to verifythe repeatability of this result. In rAAV2 and rAAV3b, VR1 amino aciddeletions had no measurable impact on vector production; however, inboth of these serotypes VR1 deletion mutations resulted in theelimination of measurable transduction in mice following intravenousinjection (FIGS. 15A-15B). Again, to verify the repeatability of theseresults, these experiments were duplicated with fresh stocks of vectorin a second cohort of mice at a 5-fold higher dose (5e11vg/injection) ofrAAV2 and rAAV2/S264del (FIG. 15C). Overall, the dramatic effectsobserved in these serotypes in response to VR1 deletion mutationssuggests that maintaining a defined VR1 structure is critical towardsthese capsids' abilities to complete a productive viral life-cycle.

Example 11 Identification of a Capsid Motif that Enables HighlyEfficient Hepatic Transduction

Previous studies have demonstrated that rAAV2 capsids harboring aminoacid insertion mutations following position 264 in the capsid protein(creating a de novo position 265) exhibit markedly enhanced transductionefficiency in skeletal muscle following intramuscular injection (Bowleset al., Mol. Ther. 20(2):443 (2012); Li et al., J. Virol. 2012.86(15):7752 (2012)). The insertion of aspartic acid in particular(rAAV2/265D) has been found to enhance transduction by orders ofmagnitude (Li et al., J. Virol. 2012. 86(15):7752 (2012)). As thedeletion of VR1 amino acids rendered rAAV2 capsids incapable oftransduction, we next examined whether the alternative—the insertion ofamino acids into VR1—could enhance the biodistribution phenotype ofrAAV2. rAAV2 and rAAV2/265D capsids packaging CBA-luc were injected intothe tail vein of mice and live animal bioluminescent imaging wasperformed at 9 dpi (FIG. 18A). At 10 dpi organs were harvested andquantified for transgene expression, as described above. Intriguingly,while VR1 amino acid deletions in other serotypes led to sharpreductions in hepatic transduction, the insertion of additional aminoacids into VR1 led to an opposite phenotype. Hepatic transduction wasincreased by 10-fold in rAAV2/265D relative to rAAV2 (FIG. 14B), whiletransduction differences between the two capsids differed by ≤2-fold inall other tissues studied (FIG. 14B and FIG. 15A). Vector genome copiesper host cell differed by less than 1-fold between the two capsids inheart and GC tissues; however, there was a 10-fold increase in vectorgenome copies per liver cell in mice administered rAAV2/265D versusrAAV2 (FIG. 18C). Thus, rAAV2/265D capsids are either more capable oftargeting to and entering liver cells than are rAAV2 capsids, or thegenomes delivered by these mutant capsids are better able to persistwithin these cells (or both).

To determine whether the presence of an aspartic acid in VR1 couldenhance hepatic transduction in additional serotypes, substitutionmutations (Table 11) were made in rAAV1 (rAAV1/T265D) and rAAV6(rAAV6/T265D), and an insertion mutation was made in rAAV3b(rAAV3b/265D). In all cases, a strong transduction preference for theliver was observed: rAAV1/T265D transduced the liver by approximately212.5-fold higher than rAAV1 (FIG. 18H), rAAV6/T265D by 28-fold relativeto rAAV6 (FIG. 18K), and rAAV3b/265D by 9-fold relative to rAAV3b (FIG.18E). In the case of rAAV1 and rAAV1/T265D, transduction differencesbetween the two constructs differed by 2.5-fold or less in all othertissues examined (FIG. 18H and FIG. 19C). rAAV6/T265D enhancedtransduction of cardiac and GC muscle relative to rAAV6 by 32- and4.5-fold, respectively (FIG. 18K). In all remaining tissues,transduction efficiencies of the two constructs were virtually identical(FIG. 19D). Finally, when comparing the biodistribution of rAAV3b andrAAV3b/265D, the mutant construct enhanced transduction by approximately3-fold in cardiac and diaphragm tissues, while no remarkabletransduction differences between the two constructs were observed in anyremaining tissues (FIG. 18E and FIG. 19B). Taken together, the data showthat the insertion or substitution of aspartic acid into VR1preferentially enhances hepatic transduction following intravenousadministration of rAAV.

TABLE 11 Variable region 1 amino acidsequences and aspartic acid insertion andsubstitution mutations made in select rAAV capsids. SerotypeVariable Region 1 Mutations     * rAAV1 ₂₆₂ SSASDGASNDNHY₂₇₃ T265D(SEQ ID NO: 1)     * rAAV2 ₂₆₂ SSQSDGAS ₂₆₈ 265D (SEQ ID NO: 2)     *rAAV3b ₂₆₂ SSQSDGASNDNHY₂₇₃ 266D (SEQ ID NO: 3)     * rAAV6 ₂₆₂SSASDGASNDNHY₂₇₃ T265D (SEQ ID NO: 6)     * rAAV8 ₂₆₃NGDSGGAT₂₇₀ T265D(SEQ ID NO: 7)      * rAAV9 ₂₆₁SNSDSGGSS₂₆₉ S265D (SEQ ID NO: 8) Boldedtext indicates conserved primary amino acid sequence; asteriskshighlight aspartic acid mutations.

To determine if transduction efficiency correlated with genome copynumber within each tissue, vector genome copies per host cell werequantified for heart, liver, and GC tissue samples. There were increasednumbers of vector genomes present in the livers of mice treated withrAAV1/T265D and rAAV3b/265D relative to wild-type counterparts (4.5-foldand 182-fold, respectively; FIGS. 18F, 18I). In the case of rAAV6,vector genome copies per liver cell were virtually identical betweenwild-type and mutant capsids (FIG. 18I). VR1 aspartic acid mutationsalso substantially enhanced the number of vector genome copies per GCcell in mice treated with rAAV1/T265D or rAAV3b/T265D (60-fold and51-fold more genomes than wild-type, respectively; FIGS. 18F, 18I, 18L),though transduction efficiencies between mutant and wild-type constructsin this tissue type were identical (in either case, values measuredwithin 2-fold of those obtained for wild-type capsids). The lack ofconsistent correlation between vector genome copy numbers andtransduction efficiencies strongly suggests that post entry processingof viral capsids is at least partially responsible for driving theenhanced liver transduction efficiencies of these mutant capsids.

While rAAV1, rAAV2, rAAV3b and rAAV6 capsids were made to contain thesame SSXSDGAS (SEQ ID NO:47) VR1 amino acid sequence following 265Dmutation, rAAV8 and rAAV9 have substantially different amino acidsequences in this region of the capsid (Table 11). Therefore, to clarifywhether the enhanced preferential liver transduction produced by capsidsbearing a 265D mutation was due to primary amino acid sequence versussome electrostatic or structural effect imparted by aspartic aciditself, 265D mutations were made in rAAV8 and rAAV9 capsids. rAAV8/T265Dand rAAV9/T265D capsids packaging CBA-luc were produced in tandem withrAAV8 and rAAV9, and all were intravenously injected into mice.Bioluminescent imaging was performed at 10 dpi. In rAAV8 and rAAV9capsids, the T265D mutation substantially reduced overall transductionefficiency relative to wild-type capsids (FIG. 20). These resultssuggest that the SSXSDGAS (SEQ ID NO:48) VR1 amino acid sequence is thedriving force behind enhanced hepatic transduction efficiency in therelevant rAAV capsids, and not a structural feature imparted by asparticacid itself.

Example 12 Impact of Capsid Heparin Binding Ability on TransductionEfficiency of VR1 Mutant Capsids

We have previously demonstrated that the ability of a given capsid tobind to heparin sulfate impedes the enhanced transduction phenotypeproduced by VR1 mutation in capsids administered via intramuscularinjection (Warischalk et al., Mol. Ther. 2015). It has been previouslyshown that heparin binding can be disrupted in rAAV2, rAAV3b, and rAAV6through single point mutations in each capsid (Kern et al., J. Virol.77(20):11072 (2003); Opie et al., J. Virol. 77(12):6995 (2003); Lerch etal., Virology, 423(1):6 (2012); Wu et al., J. Virol. 80(22):11393(2006)). Therefore, to determine whether VR1 mutant capsid transductionefficiency could be further enhanced, these mutations were incorporatedinto rAAV2, rAAV3b, and rAAV6 to create rAAV2/R585E, rAAV3b/R594A, andrAAV6/K531E. The inability of these mutant capsids to interact withheparin was verified using heparin affinity chromatography. The abovecapsids were packaged with CBA-luc and intravenously administered tomice in tandem with rAAV2/265D_R585E, rAAV3b/265D_594A, andrAAV6/T265D_K531E and their single mutant and wild-type counterparts. At10 dpi, mice were sacrificed and livers removed for quantitation oftransgene expression. As has been previously shown (Asokan et al., Nat.Biotechnol. 28(1):79 (2010); Kern et al., J. Virol. 77(20):11072(2003)), impairing heparin binding from the rAAV2 capsid decreasedhepatic transduction in mice by about 6.5-fold (FIG. 21A). However, whencombining this heparin-null rAAV2 capsid with the VR1 mutation 265D, theresultant rAAV2/265D_R585E outperformed both rAAV2 and rAAV2/265D in theliver by 132- and 11-fold, respectively (FIG. 21A). Impairing capsidheparin binding ability in rAAV3b and rAAV6 in combination with the VR1265D mutation also enhanced transduction compared to the respectiveparent capsids. rAAV3b/265D_R594A transduced the liver by 53-fold overrAAV3b and 2.5-fold over rAAV3b.265D, while rAAV6/T265D_K531E wasrespectively 46-fold and 4-fold more efficient than rAAV6 andrAAV6/T265D (FIGS. 21B, 21C). Finally, because VR1 deletion mutationsapplied to rAAV6 capsids enhanced transduction efficiency in muscletissue (unlike similar mutations in rAAV2 and rAAV3b which becametransduction deficient following VR1 amino acid deletions), we alsosought to determine whether nullification of rAAV6 heparin binding wouldserve to further increase transduction within this context. Individualmice were administered 1e11vg of rAAV6, rAAV6/T265del, rAAV6/K531E orrAAV6/T265del_K531E. At 10 dpi, mice were sacrificed and heart and GCsamples removed to determine ex vivo luciferase expression. As withrAAV6/T265D, above, abrogation of rAAV6 capsid heparin binding abilityfurther enhanced the transduction efficiency of rAAV6/T265del.rAAV6/T265del_K531E was 170-fold and 13-fold more efficient attransducing cardiac tissue than rAAV6 and rAAV6/T265del, respectively,9027-fold and 2795-fold more efficient than each in GC tissue (FIG.21D). Taken together, these studies indicate that in order to optimizethe transduction efficiency of VR1 mutant capsids, it is necessary toensure that capsid heparin binding capabilities are removed from theapplicable serotypes.

Example 13 Transduction of VR1 Mutant Capsids Relative to Most EfficientWild-Type Serotypes

When delivered intravenously into mice, rAAV8 and rAAV9 are generallyconsidered to be the most efficient serotypes at transducing hepatic andcardiac tissue, respectively (Bish et al., Hum. Gene Ther. 19(12):1359(2008); Davidoff et al., Mol. Ther. 11(6):875 (2005)). Therefore, toadequately assess the in vivo performance of our collection of VR1mutant capsids within the context of existing reagents, capsids bearingVR1 deletion mutations and those bearing VR1 insertion mutations wereevaluated in tandem with rAAV9 and rAAV8, respectively. rAAV1/T265del,rAAV6/T265del, rAAV6/T265del_K531E, rAAV7/T265del, rAAV8/T265del,rAAV9/S264del_S266del and rAAV9 capsids packaging CBA-luc were producedconcurrently and titered together in the same reaction plate via qPCR.Mice were individually injected with 1e11vg of each construct, andsacrificed at 10 dpi to collect heart and liver tissue samples forquantitative analysis. Liver tissue was collected in addition to heartas a measure of vector specificity. rAAV1/T265del, rAAV6/T265del,rAAV6/T265del_K531E, and rAAV7/T265del each transduced the heart atlevels identical to rAAV9, while rAAV8/T265del was approximately 5-foldmore efficient than rAAV9 and rAAV9/S263A_S265del approximately 3-foldless efficient (FIG. 22A). We next wanted to determine whether ourmutants preferentially transduced cardiac tissue over other organs suchas the liver. All VR1 mutant capsids exhibited greater cardiotropismthan rAAV9 (FIGS. 22B, 22C), with the exception of rAAV8/T265del, whichtransduced the liver at an equivalent level as rAAV9. rAAV6/T265delproduced the most promising cardiotropic profile, transducing the heartapproximately 582 times more efficiently than the liver (FIG. 22C)versus rAAV9, which transduced the heart only 2-fold more efficientlythan the liver. Collectively, these data present six new reagents with amore favorable cardiotropism than rAAV9.

To assess the ability of our 265D mutant capsid panel to transduce theliver relative to rAAV8, rAAV1/T265D, rAAV2/265D_R585E,rAAV3b/265D_R594A, and rAAV6/T265D_K531E vectors packaging CBA-luc wereproduced in tandem with rAAV8. All constructs were titered on the sameqPCR plate and injected into mice at a dose of 1e11vg. Mice weresacrificed at 10 dpi and samples of liver tissue removed for analysis.None of the 265D mutant capsids reached the transduction efficiency ofrAAV8, though rAAV2/265D_R585E came within <3-fold (FIG. 22D).rAAV1/T265D and rAAV6/T265D_K531E each transduced the liverapproximately 8-fold less efficiently than rAAV8, and rAAV3b/265D_R594Aby approximately 17-fold less efficiency (FIG. 22D). Collectively, theseresults show that while the insertion or substitution of an asparticacid residue into VR1 can substantially enhance hepatic transductionefficiency in numerous serotypes, rAAV8 is still superior intransduction of the liver in this mouse model.

Example 14 Cardiotropism of VR1 Mutant Capsids is Enhanced byIncorporation of Y→F Mutations

Mutation of select tyrosine residues on the rAAV capsid surface has beenshown to enhance rAAV transduction efficiency by avoiding thephosphorylation and ultimate proteasomal degradation of intracellularcapsids before they are able to reach the nucleus (Zhong et al.,Virology 381(2):194 (2008)). We wanted to determine if these mutationscould synergize with VR1 mutant capsids to enhance transductionefficiency while maintaining the biodistributive phenotypes imparted byVR1 mutation. As rAAV1 has been the most utilized serotype fortranslational human studies aimed at correcting cardiac and skeletalmuscle disease (Flotte et al., Hum. Gene Ther. 22(10):1239 (2011);Greenberg et al., JACC Heart Fail. 2(1):84 (2014)), an rAAV1 capsid wascreated to include both Y445F and T265del mutations. Thoughtyrosine-to-phenylalanine mutations have not been previously evaluatedin the context of rAAV1, Y445F has been proven to enhance transductionefficiency in rAAV6 following intramuscular injection (Qiao et al., Hum.Gene Ther. 21(10):1343 (2010)). rAAV1 and rAAV6 have >99% homology incapsid protein sequence (Wu et al., J. Virol. 80(22):11393 (2006)), thusit was reasonable to expect that this mutation may function similarly inthe context of rAAV1. Nevertheless, rAAV6 was also included in thisanalysis. Wild-type rAAV1 and rAAV6 capsids packaging CBA-luc wereproduced concurrently with single and double T265del and Y445F mutantcapsids. Mice were injected via the tail vein with 1e11vg of each, andheart, liver, and GC tissue samples were removed at 10 dpi for analysis.In both serotypes, the double T265del_Y445F mutant capsids furtherimproved cardiac transduction efficiency: rAAV1/T265del_Y445F transducedthe heart 66-fold and 9-fold higher than rAAV1 and rAAV1/T265del (FIG.23A), respectively, while rAAV6/T265del_Y445F transduced 30-fold and3-fold higher than rAAV6 and rAAV6/T265del. The double T265del_Y445Fmutation led to variable effects in GC tissue, wherein transduction wasenhanced by 3.5-fold in rAAV6/T265del_Y445F versus rAAV6/T265del (FIG.23B), and was virtually the same between the rAAV1 counterpoints tothese mutants. In both serotypes the liver detargeting imparted by theVR1 mutant capsids was wholly maintained in the context of Y445Fmutation. Curiously, the Y445F mutant alone could not enhance thetransduction efficiency of either rAAV1 or rAAV6 following intravenousdelivery, despite the fact that in cell culture experiments the mutantenhanced transduction markedly over wild-type capsids. To our knowledge,these mutants have not been previously evaluated following intravenousinjection into mice and thus it is difficult to speculate why they wouldnot outperform their parental counterparts via this route of delivery.Perhaps a tyrosine residue at position 445 is required for successfultrafficking across barriers imposed by blood vasculature. Altogether,these data suggest that incorporating Y445F into VR1 mutant capsids canfurther enhance cardiac transduction while maintaining aliver-detargeted biodistribution profile.

We present here a novel capsid engineering method that can be used toimprove the targeting and transduction of muscle tissue in systemicallydelivered rAAV. Previous observations have shown that disrupting thestability of rAAV1 capsid loop VR1 via the deletion of select aminoacids significantly enhanced skeletal muscle transduction followingintramuscular injection (Warischalk, J. K. a. S., Mol. Ther. 2015). Themutations most efficient at enhancing transduction correlated to thosethat were most disruptive to VR1 intra-loop hydrogen bond networks.These observations led us to probe whether the targeted destabilizationof VR1 in rAAV1 could induce preferential targeting of muscle tissuefollowing intravenous administration, and whether this approach could beused to engineer additional rAAV serotypes towards producing a similaroutcome. Our strategy was effective in enhancing the biodistributiveproperties of five of the nine serotypes studied. In rAAV1 and rAAV6capsids, a robust gain of function in transducing cardiac and/orskeletal muscle tissues was observed, while in rAAV7, rAAV8, and rAAV9capsids, native transduction levels were maintained in muscle tissuewhile liver transduction efficiency was dramatically decreased.Significant deleterious effects following VR1 mutation were noted in theremaining serotypes, including the inability to produce intact virionsin rAAV4 and rAAV5 and severe transduction deficiencies in rAAV2 andrAAV3b. Thus, our method provides functional insights into capsidtopology that can either be systematically incorporated or avoided whendesigning novel vectors.

Intra-protein hydrogen bond networks regulate functionality andstructural dynamics in virtually every class of proteins, includingnumerous viral species (for example, see Worth et al., BMC Evol. Biol.10:161 (2010)). However, barring the acquisition of empirical structuraldata, only correlative evidence currently supports that hydrogen bonddestabilization is the driving force behind the improved phenotypes ofthe VR1 mutant capsids created in this study. Nonetheless, the evidencedoes provide reasonable support of the hypothesis. First, the strategyworked as intended in the majority of serotypes tested. Second, rAAV6residues S262 and T265 form a hydrogen bond pair that works together tostabilize the rest of VR1 (FIG. 11F), and the individual deletion ofeither of these residues resulted in equivalent biodistributivephenotypes (FIG. 16C). Third, VR1 stability appears to depend on twodifferent sets of hydrogen bond partners in rAAV9 (FIG. 11H), and thedesired muscle-tropic biodistribution phenotype could only be obtainedfollowing mutation of both of them (FIG. 14G); individual mutations ofeither residue were ineffective (FIG. 17D). Finally, in rAAV8 only asingle hydrogen bond exists in VR1: that of the position T265 amino acidside chain hydrogen bonding to itself (FIG. 11G). While the deletion ofT265 resulted in substantial detargeting of the liver (FIG. 14E), theremoval of the neighboring S266 did not (FIG. 24). Coupled with thedestructive effects that VR1 destabilization has on rAAV2, rAAV3b, rAAV4and rAAV5, the evidence suggests that a precisely defined structure forthis capsid loop is highly influential to the productive advancement ofrAAV through its life-cycle.

While disruptions to VR1 structure clearly result in vectors withenhanced systemic properties, further details behind the mechanismgoverning these novel phenotypes remain elusive. Individual hydrogenbond pairs have been found to control capsid stability and infectiousefficiency in diverse viral species (Tipper et al., J. Virol.88(18):10289 (2014); Tso et al., J. Mol. Biol. 426(10):2112 (2014); Rueet al., J. Virol. 77(14):8009 (2003); Speir et al., J. Virol. 80(7):3582(2006)) and have additionally been hypothesized to modulate rAAVassembly (Grieger et al., J. Virol. 80(11):5199 (2006)). However, anestablished heat dissociation protocol designed to measure rAAV capsidstability (Horowitz et al, J. Virol. 87(6):2994 (2013)) revealed nodifferences between wild-type and VR1 mutant capsids. Furthermore, asimple increase in capsid dissociability seems unlikely to result inbroad changes to vector biodistribution. The VR1 loop of rAAV lies at aninterface between monomeric subunits on the exposed capsid surface andcan therefore potentially interact with additional proteins.Intra-capsid hydrogen bond networks are known to modulate capsidinteractions with binding partners, such as during glycan catalysis byinfluenza neuraminidase (von Grafenstein et al., J. Biomol. Struct. Dyn.33(1):104 (2015)), receptor engagement by human polyomavirus (Khan etal., J. Virol. 88(11):6100 (2014)), and evasion of the anti-viral factorTRIMS a by HIV-2 (Miyamoto et al., PLoS One 6(7):e22779 (2011)). VR1mutation may therefore alter capsid stability so as to prevent capsidinteractions with a preferred cell surface receptor, enabling analternate entryway into the nucleus. The consistent reduction in hepaticvector genome populations of mice administered VR1 deletion mutantcapsids supports this notion, as does the finding that vector genomesdelivered to cardiac cells by mutant capsids were far more efficientlyexpressed than those delivered by wild-type counterparts. Furthermore,this concept is not without precedent, as rAAV2 exhibits improved muscletargeting and decreased liver transduction when the capsid is mutated toeliminate heparin binding (Asokan et al., Nat. Biotechnol. 28(1):79(2010); Kern et al., J. Virol. 77(20):11072 (2003)). rAAV9 also exhibitsa liver-detargeted, systemically enhanced phenotype when mutated toreduce galactose binding (Shen et al., J. Virol. 86(19):10408 (2012)).It is notable that VR1 mutations have been shown to reduce the abilityof rAAV2 and rAAV6 capsids to bind to heparin sulfate (Warischalk etal., Mol. Ther. 2015). Additionally, the N-terminal region of VR1 ispositioned on the capsid in close enough proximity (<3 A) to thegalactose-binding footprint of rAAV9 that direct interactions betweenthe two regions are feasible (Bell et al., J. Virol. 86(13):7326(2012)). Such observations bring up intriguing questions as to whetherreceptors identified for different rAAV serotypes in vitro are potentialartifacts from passaging through cell culture, and whether rAAV couldhave a far greater in vivo transduction potential than previouslybelieved if such artifacts are mutationally ablated.

From a structural perspective, the observation that VR1 deletionmutations render rAAV4 and rAAV5 incapable of producing intact virionsis straightforward. In both of these serotypes, VR1 forms a relativelyshort loop that is primarily hydrogen bonded to underlying B-sheets.Therefore, disrupting the stability of this region of the capsid couldconceivably result in gross structural changes that prevent capsidmonomers from interacting with each other. Understanding why VR1deletion mutations render rAAV2 and rAAV3b as transduction deficient isless clear. Despite being unable to transduce mouse tissues,rAAV2/S264del and rAAV3b/S262del are able to transduce cells in culture.Therefore, any deficiencies in the transduction of mouse tissues stemsfrom some facet of the in vivo environment. Future experiments aim todissect these observations further. The failure to improve rAAV2 throughVR1 amino acid deletions was not without merit, however, as it led tothe serendipitous discovery that insertions of aspartic acid into VR1produce the opposite phenotype of deletion mutations: robust enhancementof hepatic transduction following systemic administration of vector. Themechanistic details governing the behavior of VR1 insertion mutants arecurrently unknown. Aspartic acid is a known phosphomimetic and couldtherefore potentially redirect capsid trafficking within the cell.Indeed, serine and threonine phosphorylation has been suggested to tagrAAV capsids for proteasomal degradation (Gabriel et al., Hum. GeneTher. Meth. 24(2):80 (2013)). However, open source phosphorylation-siteprediction software (NetPhos2.0) does not indicate VR1 (either modifiedor unmodified) as a site of phosphorylation, and data from cell cultureexperiments utilizing proteasome inhibitor do not support that thephenotype of 265D mutant capsids is a result of reduced proteasomalclearance. Another possibility is that aspartic acid insertion into VR1produces the opposite effect of amino acid deletion: stabilizing thestructure of VR1 as opposed to destroying it. Aspartic acid residues arewell known for their stabilizing effect on protein structure throughparticipation in intra-protein interactions such as carbonyl stacking(Deane et al., Protein Eng. 12(12):1025 (1999)), salt bridges (Speare etal., J. Biol. Chem. 278(14):12522 (2003)), and hydrogen bond networks(Worth et al., BMC Evol. Biol. 10:161 (2010)). If this were true, it isconceivable that while VR1 deletion mutations destabilize this region ofthe capsid so as to prevent interaction with a given receptor, asparticacid insertions into VR1 instead enhance both capsid structuralstability and resultant protein:protein interactions. The majority of265D mutant capsids increased vector genome populations in both theliver and the GC, suggesting that the 265D mutation enhances cell entry.However, while this appears advantageous in the case of hepatictransduction, transduction efficiency in GC was not enhanced in thecontext of the 265D mutation, suggesting that post attachment processingof the capsid is also being affected by the mutation.

In summary, our studies offer new structural insights into the influenceof the VR1 region of the rAAV capsid on transduction efficiency andtissue targeting in vivo. These insights allowed the development of twoconserved rational engineering strategies that improve tissue targetingfollowing systemic administration of vector. At first glance, the VR1deletion mutant capsid collection appears to be the more clinicallyrelevant, as six novel vectors were developed that can perform at orabove the level of rAAV9 at targeting the heart. While this is indeed avaluable advancement in rAAV vectorology, the 265D mutant capsidcollection should not be overlooked in terms of clinical value. ThoughrAAV8 is consistently considered the best hepatic transducer in mousemodels (Davidoff et al., Mol. Ther. 11(6):875 (2005)), and though the265D capsid collection could not outperform rAAV8 in our study, it isalso notable that rAAV8 may not be the most ideal vector fortranslational human gene therapy applications. Indeed, both rAAV2 and avariant of rAAV3b have been found to infect human hepatocytes withhigher efficiency than rAAV8 (Lisowski et al., Nature, 506(7488):382(2014)). Thus, both of our VR1 mutant capsid collections have thecapacity to expand the reagent pool available for either muscle- orliver-oriented gene therapy applications. It is our hope that thisexpansion enables better personalization of gene therapy moving forward,perhaps allowing a patient's preexisting neutralizing antibody profile(Louis et al., Hum. Gene Ther. Meth. 24(2):59 (2013)) to aid in choosingthe best-suited serotype for their individual needs and enhancing theability to readminister vector if necessary.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A modified adeno-associated virus (AAV)capsid protein comprising a deletion and/or substitution of one or moreamino acids in the variable region 1 (VR1) loop, wherein the VR1 loopcorresponds to amino acids that fall at positions 261 to 273 from thestarting methionine of the AAV1 VP1 capsid protein, excluding AAV2,AAV3b, AAV4, and AAV5, wherein the deletion and/or substitution causesregional destabilization within the loop due to the targeted destructionof hydrogen bonding patterns orchestrated by the residues, wherein themodified AAV capsid protein confers increased transduction efficiencyand/or modified tissue specificity to an AAV vector relative to an AAVvector comprising an otherwise identical capsid protein that does notcontain the deletion and/or substitution.
 2. The modified AAV capsidprotein of claim 1, wherein one or more of amino acid residuescorresponding to 261 to 273 of AAV1 VP1 capsid protein or thecorresponding amino acid residues from another AAV capsid protein aredeleted.
 3. The modified AAV capsid protein of claim 1, wherein one ormore of amino acid residues 261 to 269 of AAV1 VP1 capsid protein or thecorresponding amino acid residues from another AAV capsid protein aredeleted.
 4. The modified AAV capsid protein of claim 1, wherein aminoacid residue 265 of AAV1 VP1 capsid protein or the corresponding aminoacid residue from another AAV capsid protein is deleted.
 5. The modifiedAAV capsid protein of claim 1, wherein the capsid protein comprises anamino acid sequence from an AAV serotype that binds to heparin sulfate,wherein one or more amino acid residues that mediate binding of thecapsid protein to heparin sulfate are substituted and/or deleted,wherein binding of the capsid protein to heparin sulfate issubstantially reduced.
 6. The modified AAV capsid protein of claim 5,wherein the capsid protein comprises the amino acid sequence from anAAV3a, AAV6, or AAV8 serotype.
 7. The modified AAV capsid protein ofclaim 5, wherein the capsid protein comprises the amino acid sequencefrom an AAV6 serotype, wherein amino acid residue 531 (VP1 numbering)has been substituted to substantially reduce binding to heparin sulfate.8. The modified AAV capsid protein of claim 7, wherein amino acidresidue 531 (VP1 numbering) has been substituted with glutamic acid. 9.A polynucleotide encoding the modified AAV capsid protein of claim 1.10. An adenovirus-associated virus (AAV) capsid comprising the modifiedAAV capsid protein of claim
 1. 11. An adenovirus-associated virus (AAV)vector comprising: (a) the AAV capsid of claim 10; and (b) a nucleicacid comprising a recombinant viral template, wherein the nucleic acidis encapsidated by the AAV capsid.
 12. The AAV vector of claim 11, whichis selected from the group consisting of AAV1, AAV3a, AAV6, AAV7, AAV8,and AAV9.
 13. A pharmaceutical composition comprising the AAV vector ofclaim 11 in a pharmaceutically acceptable carrier.
 14. A method ofdelivering a nucleic fid to a cell, the method comprising contacting thecell with the AAV vector of claim 11 under conditions sufficient for thenucleic acid to enter the cell.
 15. A method of delivering a nucleicacid to a subject, the method comprising administering to the subjectthe AAV vector of claim
 11. 16. A method of delivering a nucleic acid toa subject, the method comprising administering to the subject a cellthat has been contacted with the AAV vector of claim 11 under conditionssufficient for the nucleic acid to enter the cell.
 17. A method ofproducing a recombinant adenovirus-associated virus (AAV) particle,comprising providing to a cell permissive for AAV replication: (a) arecombinant AAV template comprising (i) a heterologous nucleic acid, and(ii) at least one inverted terminal repeat; and (b) a polynucleotidecomprising replication protein coding sequence(s) and sequence(s)encoding the modified AAV capsid protein of claim 1; under conditionssufficient for the replication and packaging of the recombinant AAVtemplate; whereby recombinant AAV particles are produced in the cell.18. The modified AAV capsid protein of claim 1, wherein the capsidprotein comprises the amino acid sequence from AAV1 and the deletionand/or substitution is at position T265, A263, S264, or G266 (VP1numbering).
 19. The modified AAV capsid protein of claim 1, wherein thecapsid protein comprises the amino acid sequence from AAV6 and there isa deletion and/or substitution at one or more positions selected fromthe group consisting of S262, S264, T265, and H272 (VP1 numbering). 20.The modified AAV capsid protein of claim 1, wherein the capsid proteincomprises the amino acid sequence from AAV8 and there is a deletionand/or substitution at T265 (VP1 numbering).
 21. The modified AAV capsidprotein of claim 1, wherein the capsid protein comprises the amino acidsequence from AAV9 and there is a deletion and/or substitution at one ormore positions selected from the group consisting of S263, S265, andG267 (VP1 numbering).